Compositions and methods for CD20 immunotherapy

ABSTRACT

The present disclosure provides compositions and uses thereof for treating a disease or disorder associated with CD20 expression. Treatments of this disclosure include use of a host cell expressing a fusion protein, such as an anti-CD20 CAR, optionally in combination with a CD20-specific binding molecule, a chemotherapeutic, an inhibitor of an immunosuppression component, or combinations thereof.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. patent applicationSer. No. 16/086,290, filed Sep. 18, 2018 (now allowed), which is a 371national stage application of International Patent Application No.PCT/US2017/023098, filed Mar. 17, 2017, which claims the benefit of U.S.Provisional Application No. 62/320,327, filed Apr. 8, 2016, and U.S.Provisional Application No. 62/310,541, filed Mar. 18, 2016, whichapplications are incorporated herein by reference in their entirety.

STATEMENT OF GOVERNMENT INTEREST

This invention was made with government support under CA154874 awardedby the National Institutes of Health. The government has certain rightsin the invention.

STATEMENT REGARDING SEQUENCE LISTING

The Sequence Listing associated with this application is provided intext format in lieu of a paper copy, and is hereby incorporated byreference into the specification. The name of the text file containingthe Sequence Listing is 360056_441D1_SEQUENCE_LISTING.txt. The text fileis 213 KB, was created on Nov. 24, 2020, and is being submittedelectronically via EFS-Web.

BACKGROUND

Adoptive transfer of genetically modified T cells has emerged as apotent therapy for various malignancies. The most widely employedstrategy has been infusion of patient-derived T cells expressingchimeric antigen receptors (CARs) targeting tumor-associated antigens.This approach has numerous theoretical advantages, including the abilityto target T cells to any cell surface antigen, circumvent loss of majorhistocompatibility complex as a tumor escape mechanism, and employ asingle vector construct to treat any patient, regardless of humanleukocyte antigen haplotype. For example, CAR clinical trials for B-cellnon-Hodgkin's lymphoma (NHL) have, to date, targeted CD19, CD20, or CD22antigens that are expressed on malignant lymphoid cells as well as onnormal B cells (Brentjens et al., Sci Transl Med 2013; 5(177):177ra38;Haso et al., Blood 2013; 121(7):1165-74; James et al., J Immunol 2008;180(10):7028-38; Kalos et al., Sci Transl Med 2011; 3(95):95ra73;Kochenderfer et al., J Clin Oncol 2015; 33(6):540-9; Lee et al., Lancet2015; 385(9967):517-28; Porter et al., Sci Transl Med 2015;7(303):303ra139; Savoldo et al., J Clin Invest 2011; 121(5):1822-6; Tillet al., Blood 2008; 112(6):2261-71; Till et al., Blood 2012;119(17):3940-50; Coiffier et al., N Engl J Med 2002; 346(4):235-42).Most investigators studying therapies for lymphoid malignancies havechosen to target CD19 since this molecule is expressed from earlierstages of B-cell differentiation than CD20 or CD22. CAR T cellstargeting CD19 can therefore be used to treat a slightly wider range ofB-cell malignancies, including acute lymphoblastic leukemia, whicharises at the pro- or pre-B cell stage of differentiation.

CD20 remains an appealing antigen, however, due to its extensiveclinical record as a successful immunotherapy target, as demonstrated intrials using rituximab, a monoclonal antibody targeting CD20 (Coiffieret al., N Engl J Med 2002; 346(4):235-42; Lenz et al., J Clin Oncol2005; 23(9):1984-92; Marcus R, et al., J Clin Oncol 2008;26(28):4579-86; Pfreundschuh et al., Lancet Oncol 2011; 12(11):1013-22).In contrast to CD19, which is readily internalized upon antibody binding(Pulczynski et al., Blood 1993; 81(6):1549-57), CD20 undergoesendocytosis much more slowly after antibody binding (Press et al., Blood1994; 83(5):1390-7; Pulczynski et al., Leuk Res 1994; 18(7):541-52).This stability could theoretically impact the quality of theimmunological synapse and subsequent CAR triggering and T cellactivation. Loss of CD19 expression on tumor cells has been described asan escape mechanism in patients treated with CD19-targeted T cells(Grupp et al., N Engl J Med 2013; 368(16):1509-18). Although CD20 losshas also been described following anti-CD20 antibody therapy,CD20-specific CAR T cells provide an alternative target that would allowsequential therapy, or could be used in concert with CD19 CART cells totarget multiple antigens simultaneously, reducing the risk of immuneescape by antigen loss.

One potential limitation of CD20 as a target antigen for CARs is thatpatients with relapsed or refractory lymphoma who are likely to becandidates for CAR T cell therapy trials will often have been treatedrecently with rituximab-containing regimens. Since antibody can persistin the serum for months, residual rituximab could theoretically blockthe binding of CARs to CD20 and prevent or weaken T-cell activation,potentially rendering therapy ineffective. In previous CD20 CAR T celltrials (Till et al., Blood 112:2261-71, 2008; Till et al., Blood119:3940-50, 2012), eligibility criteria excluded patients recentlytreated with rituximab. However, this approach significantly impactsaccrual and would ultimately limit the availability of this therapy forpatients most in need of novel treatment options.

Currently, there remains a need in the immunotherapy field forcompositions and methods for additional or alternative immunotherapiesdirected against various diseases, including cancer (e.g., leukemia,lymphoma). Presently disclosed embodiments address this need and provideother related advantages.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B show schematic diagrams of CD20-specific CAR constructscontaining scFvs from different anti-CD20 antibodies (Leu16, 1F5, and1.5.3). (A) Shows CD20-specific CAR constructs and their respectivemature CAR proteins. (B) Shows additional mature CD20-specific CARproteins.

FIGS. 2A-2F show rituximab and ofatumumab block antigen binding ofantibody used to generate a CAR scFv. Ramos cells (CD20⁺) were incubatedwith the indicated rituximab (A-C) or ofatumumab (D-F) concentrationsfor 30 minutes, followed by incubation with PE-labeled anti-CD20antibody (clone Leu16) or isotype control at either 4° C. (A and D) or37° C. (B and E) for 30 minutes. Cells were washed and analyzed by flowcytometry to determine available CD20 binding sites as measured by PEfluorescence intensity. The graphs depicted in FIG. 2C and FIG. 2Fsummarize the geometric mean fluorescence intensity (MFI) at either 4°C. or 37° C. as a function of rituximab or ofatumumab concentration,respectively. The data are representative of three independentexperiments.

FIGS. 3A and 3B show the effect of rituximab on CAR T cell function invitro. The indicated B-cell NHL cell lines were irradiated and incubatedfor 30 minutes at room temperature with varying rituximab concentrations(at 2× the concentrations during incubation to yield the indicated finalconcentrations after addition of T cells). CFSE-stained T cellsexpressing the Leu16-28-BB-z-tEGFR CD20-specific CAR were added to thetarget cells at a 1:1 volume and ratio. (A) Proliferation of the T cellswas analyzed 4 days later by flow cytometry for CFSE dilution. Thepercent divided CD3⁺ T cells relative to unstimulated T cells are shownon the left axis (filled bars). Cell size of CD3⁺ T cells as determinedby geometric mean of forward scatter (subtracting size of cells in mediaonly) is shown on the right axis (open bars). (B) Cytokine secretion ofthese T cells was measured by Luminex assay using supernatants from 24hours after restimulation. Interleukin (IL)-2 concentrations are shownon the left y-axis and Interferon (IFN)-γ and tumor necrosis factor(TNF)-α on the right y-axis. The data shown are representative of 3independent experiments.

FIG. 4 shows the effect of rituximab on CAR T cell-mediatedcytotoxicity. The indicated ⁵¹Cr-labeled target cells were pre-incubatedfor 30 minutes with rituximab (at 2× the concentrations duringincubation to yield the indicated final concentrations after addition ofT cells), and then CD8⁺ T cells expressing the Leu16-28-z CAR were addedat the E:T ratios shown in a standard 5-hour ⁵¹chromium-release assay.Mock-transduced T cells, and samples with rituximab and target cellsonly (“0:1”) were used as negative controls. The average value ofduplicate wells is shown, with error bars representing standarddeviation. The data are representative of results from 4 independentexperiments.

FIGS. 5A-5C show that sensitivity to rituximab blockade is dependent onCD20 antigen density on target cells. K562 cells transduced with CD80and CD20 (“K80-20”) were cloned by limiting dilution, selected for high,medium, or low levels of CD20 expression (FIG. 10 ), and used as targetcells in assays for (FIG. 5A) proliferation and cell size (geometricmean forward scatter of gated CD3⁺ cells minus the size of cells inmedia only) using CFSE-labeled Leu16-28-z CAR-transduced T cells asdescribed in FIG. 3 ; (FIG. 5B) cytokine secretion of the Leu16-28-zCAR-transduced T cells at 24 hours from (FIG. 5A) above, measured byLuminex assay; and (FIG. 5C) cytotoxicity using Leu16-28-zCAR-transduced CD8⁺ T cells by ⁵¹Cr-release assay as described in FIG. 4. Data are representative of three independent experiments. Absolutevalues for cytokine secretion are shown in FIG. 11 .

FIGS. 6A and 6B show proliferation and cytokine secretion by T cellsexpressing an anti-CD20 CAR. Healthy donor T cells were sorted andstimulated using anti-CD3/28 beads, followed by transduction withlentiviral vector encoding the 1F5-28-BB-z CAR construct. (A) At day 9after stimulation, CAR T cells were labeled with CFSE, restimulated witheither K562-CD80-CD20 (“K80-20”) or K562-CD80 (“K80”) target cells thathad been irradiated, and CFSE dilution was measured by flow cytometry 4days later to measure T cell proliferation. The percent divided CD3⁺ Tcells relative to unstimulated T cells are shown. Mock-transduced Tcells were used as a negative control. (B) Cytokine secretion by the Tcells was determined by harvesting supernatant samples from the abovecultures at 24 hours after restimulation and analyzing the indicatedcytokine concentration by Luminex assay.

FIGS. 7A-7E show the in vivo effect of rituximab on CD20 CAR T cellfunction. Nod-SCID-γ^(−/−) (NSG) mice were injected intravenously (i.v.)with 5×10⁵ rituximab-refractory Raji-ffLuc lymphoma cells, followed byone of the following treatments: no treatment, rituximab only (25 μg or200 μg) intraperitoneally (i.p.) 5 days later, 10⁷ 1.5.3-NQ-28-BBz CARTcells only 6 days after tumor, or rituximab (25 μg or 200 μg) i.p. at 5days followed by 10⁷ CAR T cells at 6 days after tumor. Mice were imagedtwice weekly for bioluminescence. (A) Schema of mouse experiment. (B)Average tumor burden per group over time as measured by total bodybioluminescence. The geometric mean luminescence values with 95%confidence intervals are shown, and to prevent misleading fluctuationsin tumor volume graphs, the last bioluminescence level of each mouse wascarried forward after it was killed until no mice in that groupremained. Individual bioluminescence traces are shown in FIG. 13 . (C)Kaplan-Meier plot showing overall survival of each treatment group. (D)Serum rituximab levels on the day of T cell infusion (day 6) and 1 weekpost T cell infusion (day 13). The horizontal bars denote the medianvalues. (E) Serum rituximab levels from lymphoma patients who underwentrituximab-containing salvage chemotherapy within the 4 preceding months.The gray horizontal bar line indicates the median, and black horizontalbar lines indicate the interquartile range (25-75%).

FIGS. 8A-8C show the effect of ofatumumab on CD20 CAR T cell function invitro. Irradiated Rec-1 or Raji-ffLuc cells or non-irradiated⁵¹Cr-labeled Rec-1 cells were pre-incubated for 30 minutes with 2× theindicated concentrations of ofatumumab, followed by experiments todetermine function of T cells expressing the 1.5.3-NQ-28-BB-z CAR, usingthe methodologies described in the legend of FIGS. 3 and 4 . (A) Thepercent divided CD3⁺ T cells relative to unstimulated T cells are shownon the left axis (filled bars). Cell size of CD3⁺ T cells as determinedby geometric mean of forward scatter (subtracting size of cells in mediaonly) is shown on the right axis (open bars). (B) Cytokine secretion ofthese T cells was measured by Luminex assay using supernatants from 24hours after restimulation. IL-2 concentrations are shown on the lefty-axis and IFN-γ on the right y-axis. (C) Cytotoxicity of1.5.3-NQ-28-BB-z CART cells was determined using a standard 4-hour⁵¹Cr-release assay with Rec-1 target cells. The average value ofduplicate wells is shown, with error bars representing standarddeviation.

FIG. 9 shows CD20 expression of K80-20^(low), K80-20^(med),K80-20^(high) as determined by flow cytometry. Open histograms representcells stained with FITC-conjugated 1F5 antibody (anti-CD20), and filledhistograms represent cells stained with an isotype control antibody Ab.

FIG. 10 shows the absolute cytokine concentrations from T cellsupernatants from the experiment in FIG. 5 are shown.

FIGS. 11A-11E show proliferation, cytokine secretion, and cytotoxicityof CAR T cells with fully human anti-CD20 scFv. Healthy donorCD14⁻CD45RA⁻CD62L⁺ central memory T cells were stimulated usinganti-CD3/28 beads, followed by transduction with lentiviral vectorencoding either the 1.5.3-NQ-28-z or 1.5.3-NQ-28-BB-z CAR. CAR T cellswere labeled with CFSE and restimulated with irradiated Raji-ffLuc,rituximab-refractory Raji-ffLuc (RR-Raji), or Rec-1 target cells. (A)Proliferation of 1.5.3-NQ-28-z T cells was assessed by analyzing thecells 4 days later by flow cytometry for CFSE dilution. The percentdivided CD3⁺ T cells relative to unstimulated T cells are shown. (B)Cytokine secretion by 1.5.3-NQ-28-z T cells was determined by harvestingsupernatant samples from the above cultures at 24 hours afterrestimulation and analyzing the indicated cytokine concentrations byLuminex assay. IL-2 and TNF-α concentrations are shown on the lefty-axis and IFN-γ concentrations are plotted on the right y-axis. (C)Cytokine secretion by 1.5.3-NQ-28-BB-z T cells determined as in part Babove. (D) Cytotoxicity of 1.5.3-NQ-28-BB-z CART cells was determinedusing a standard 5-hour Chromium⁵¹-release assay with the indicatedtarget cell lines. (E) Cytokine secretion by 1.5.3-NQ-28-BB-z T cells asin part (A) above and stimulated with Granta, Rec-1, FL-18, or K80-20cells.

FIG. 12 shows that rituximab-refractory Raji-ffLuc have the same CD20expression as parental Raji-ffLuc cells. Raji-ffLuc (solid-linehistogram) or rituximab-refractory Raji-ffLuc (dashed-line histogram)cells were stained with anti-CD20-PE or anti-CD20-APC antibodies andthen analyzed by flow cytometry. CD20 expression relative to isotypecontrol antibody (filled histogram) is shown for each cell line.

FIGS. 13A and 13B show bioluminescent traces and images from a xenografttumor mouse model from FIG. 7 treated with anti-CD20 CAR T cells. (A)Individual mouse bioluminescent tumor burden traces over time. Each linerepresents an individual mouse. The grey line represents a mouse with notumor, which defines the baseline autofluorescence. (B) Representativemouse bioluminescence images.

FIGS. 14A-14C show presence of circulating T cells in mice. Peripheralblood mononuclear cells (PBMC) were isolated from retroorbital bloodsamples taken at day 28 after tumor injection and analyzed by flowcytometry for human CD3, mouse CD45, and human CD19 (as a marker oftransduced T cells). (A) Representative dot plots of circulating human Tcells (gated on viable lymphocytes) are shown in left panels and CAR⁺cells (based on CD19 expression), gated on human CD3⁺ T cells are shownin right panels. (B) Summary of T cell persistence at day 28. (C)Summary CAR expression on persisting T cells. For both FIG. 14B and FIG.14C, the difference between CAR only and CAR+rituximab groups were notstatistically significant, based on unpaired two-tailed t test.

FIGS. 15A-15D show cytokine secretion by various CAR constructs invitro. Central memory (CD14⁻CD45RA⁻CD62L⁺) T cells were stimulated withanti-CD3/CD28 antibody coated beads, transduced 24 hours later withlentiviral vectors encoding the indicated CAR constructs, and expandedin vivo. At day 14, the cells were re-stimulated with either irradiatedRaji-ffLuc cells (FIG. 15A and FIG. 15C), Granta-519 cells (FIG. 15B),and Jeko cells (FIG. 15D). The “19-BB-z” construct is a clinical-gradeCD19-targeted CAR being used in clinical trials and is provided as apositive control. Supernatants were harvested 24 hours later andanalyzed by Luminex assay for interferon (IFN)-γ, IL-2, and tumornecrosis factor-α levels.

FIGS. 16A and 16B show cytokine secretion by CD20 CAR T cells. (A) CD4⁺and CD8⁺ T cells transduced with the 1.5.3-NQ-28-BB-z lentiviral vectorand expanded ex vivo were restimulated with irradiated Raji-ffLuc CD20⁺lymphoma cells. Secretion of the indicated cytokines was measured incell supernatants after 24 hours by Luminex assay. (B) CryopreservedCD4⁺ and CD8⁺ CD20 CAR T cells were thawed and restimulated with K562cells or K562 cells expressing CD20 and at 24 hours were analyzed byintracellular staining for IFN-γ by flow cytometry.

FIGS. 17A and 17B shows in vitro cytotoxicity of various CAR constructs.Central memory (CD14⁻CD45RA⁻CD62L⁺) T cells were stimulated withanti-CD3/CD28 antibody coated beads, transduced 24 hours later withlentiviral vectors encoding the indicated CAR constructs, and expandedin vivo. At day 14, the cells were used as effectors in a standard4-hour ⁵¹Cr-release assay, using (FIGS. 17A and 17B) Raji-ffLuc, and(FIG. 17B) Jeko cells as targets. The “19-BB-z” construct is aclinical-grade CD19-targeted CAR being used in clinical trials and isprovided as a positive control. The specific target cell lysis of eachCART cell population is shown.

FIGS. 18A and 18B show proliferation of CD20 CAR T cells. CD8⁺ T cellswere transduced with the 1.5.3-NQ-28-BB-z lentiviral vector (or weremock-transduced) and expanded ex vivo, and then cryopreserved. The cellswere then thawed, stained with carboxyfluorescein succinamidyl ester(CFSE), and restimulated with irradiated CD20⁺ Raji-ffLuc lymphomacells, K562 cells, or K562 cells expressing CD20. Cells were analyzed byflow cytometry 4 days later. (A) CFSE dilution of CAR⁻ cells (gated onCD3⁺/tCD19⁺) is shown. The dashed-line histogram shows CFSE fluorescenceof T cells in culture medium only, and solid-line histograms are T cellsco-incubated with target cells. (B) The percentage of divided cells isshown for each group.

FIGS. 19A and 19B show in vivo anti-tumor activity of various CARconstructs. Central memory (CD14⁻CD45RA⁻CD62L⁺) T cells were stimulatedwith anti-CD3/CD28 antibody coated beads, transduced 24 hours later withlentiviral vectors encoding the indicated CAR constructs, and expandedin vitro. The “19-BB-z” construct is a clinical-grade CD19-targeted CARbeing used in clinical trials at our center and provided as a benchmarkcontrol. NSG mice were injected i.v. with Raji-ffLuc tumor cells,followed 2 days later by i.v. injection of expanded central memory(CD14⁻CD45RA⁻CD62L⁺) T cells transduced with the 1.5.3-NQ-28-BB-z CAR,1.5.3-NQ-28-z CAR, JCAR-014 (anti-CD19-41BB-ζ) or an empty vector. (A)Tumor burden over time as assessed by bioluminescence imaging; and (B)Kaplan-Meier plot of overall survival.

FIG. 20 shows in vivo activity of CD20 CAR T cells against mantle celllymphoma. CD4⁺ and CD8⁺ CD20 CART cells were transduced with the1.5.3-NQ-28-BBz CAR and used to treat NSG mice that had been inoculated7 days earlier with Granta-ffLuc mantle cell lymphoma cells by tailvein. Kaplan-Meier plot of overall survival.

FIGS. 21A and 21B show in vivo CAR T cell persistence. Retroorbitalblood samples were obtained at serial time points after infusion ofeither CD20 CAR T cells or empty vector tCD19-expressing T cells in NSGmice bearing Raji-ffLuc disseminated tumors. CD20 CAR T cells expressingthe tCD19 transduction marker were quantified by flow cytometry at eachtime point as human CD3⁺/mouse CD45-negative/human CD19⁺ cells. (FIG.21A) tCD19⁺ T cells at 3 post-infusion time points as a percentage oftotal nucleated cells in the blood are shown (n=9 initially in CAR Tcell group). Truncated CD19⁺ cells from an empty vector mouse are shownfor reference. (FIG. 21B) In a separate experiment, the tCD19⁺ cellsfrom 2 mice in each group (empty vector vs CAR T cells) are shownlongitudinally with weekly measurements.

FIGS. 22A and 22B show comparative data for various constructs havingspacers of varying lengths. Central memory (CD14⁻CD45RA⁻CD62L⁺) T cellswere stimulated with anti-CD3/CD28 antibody coated beads, transduced 24hours later with lentiviral vectors encoding the indicated CARconstructs, and expanded in vitro. The 1F5-28-BB-z, IgG1mut havefull-length spacers. The No Linker is nearly full length but missing a 6amino acid linker (junction amino acid), and the CH3 has a truncatedspacer, missing the junction amino acids and CH2 domain. (A) On day 20,the cells were re-stimulated with Granta or Rec-1 lymphoma cells, and 24hours later supernatants were harvested and analyzed by Luminex assayfor IL-2 (right) and IFN-γ (left) concentrations. (B) Central memory Tcells (CD14⁻CD45RA⁻CD62L⁺) were stimulated with anti-CD3/anti-CD28antibody coated beads, transduced 24 hours later with lentiviral vectorsencoding the indicated CAR constructs, and expanded in vitro. The“IgG1mut NQ” has a full length CH2CH3 spacer, CH3 only is intermediatelength as discussed above, and Leu16 short lacks both CH2 and CH3domains. On day 20, cells were used as effector cells in a standard4-hour ⁵¹Cr-release assay, using Raji cells as targets.

FIGS. 23A-23F show comparative data for various constructs havingspacers with various modifications. A schematic diagram of a CAR withIgG2 junction amino acids (denoted “IgG1mut”) and N297Q (denoted “NQ”)mutations is shown (FIG. 23A). T cells expressing CARs with a wild-typeIgG1 spacer, IgG1 mutant spacer (IgG1 junction amino acids replaced withIgG2 junction amino acids), or no junction amino acids (IgG1 junctionamino acids deleted) were stained with biotinylated soluble CD64 (FcγRI)followed by streptavidin-PE and then analyzed by flow cytometry,demonstrating Fc receptor binding to wild-type but not modified spacers(FIG. 23B). T cells expressing the indicated CAR constructs wereco-incubated with K562 cells expressing CD64 (FcγRI) or parental K562lacking Fc receptors. At 24 hours after co-incubation the T cells wereevaluated for CD25 and CD69 expression by flow cytometry as anindication of activation. Dot plots represent CD3⁺CD19⁺ cells (CAR⁺ Tcells). Binding of wild type spacers to Fc receptors led to T cellactivation whereas modified spacers did not (FIG. 23C). Central memory(CD14⁻CD45RA⁻CD62L⁺) T cells were stimulated with anti-CD3/CD28 antibodycoated beads, transduced 24 hours later with lentiviral vectors encodingthe indicated CAR constructs, expanded in vitro, and injected into NSGmice 2 days after i.v. administration of Raji-ffLuc cells. (D and E)Tumor burden data by bioluminescence for two different experiments. (F)Kaplan-Meier survival curve from experiment in part (E) above.

FIG. 24 shows a diagram of a treatment schema for a clinical trialinvolving immunotherapy methods and compositions of the presentdisclosure.

FIGS. 25A and 25B show a diagram of a method of formulation and model ofadministration of anti-CD20 CAR T cells in a clinical trial.

DETAILED DESCRIPTION

The instant disclosure provides compositions and methods for reducingthe number of CD20-expressing cells or treating a disease or disorderassociated with CD20 expression (e.g., reducing the number of B-cells ortreating a disease or disorder associated with aberrant B cellactivity), comprising treating a subject with a therapeuticallyeffective amount of a host cell comprising a heterologous nucleic acidmolecule encoding a fusion protein, the fusion protein comprising anextracellular component and an intracellular component connected to theextracellular component by a hydrophobic portion, wherein theextracellular component comprises a binding domain that specificallybinds CD20 and the intracellular component comprises an effector domain.Optionally, the method may further comprise a therapeutically effectiveamount of a CD20-specific binding molecule in combination with the hostcell expressing the fusion protein specific for CD20. In certainembodiments, the fusion protein is a chimeric antigen receptor (CAR). Instill further embodiments, the CAR comprises a scFv from an anti-CD20antibody or a scTCR from a TCR specific for a CD20 antigen.

By way of background, it is generally believed that residual anti-CD20antibody levels might represent a major constraint for CD20-targeted CART cells. For example, previous studies with other targets havedemonstrated that cytokine secretion and cytotoxicity of CAR T cellstargeting carcinoembryonic antigen, Lewis-Y antigen, or CD30 are largelyunimpaired in the presence of levels of soluble cognate antigen of up to10 μg/ml (Hombach et al., Gene Ther 2000; 7(12):1067-75; Hombach et al.,Gene Ther 1999; 6(2):300-4; Nolan et al., Clin Cancer Res 1999;5(12):3928-41; Westwood et al., J Immunother 2009; 32(3):292-301); itwas observed that levels higher than this are potentially inhibitory(Hombach et al., Gene Ther 2000; 7(12):1067-75). In this disclosure, itwas surprisingly found that various anti-CD20 antibodies (e.g.,rituximab) in clinically relevant concentrations largely did not affectthe activity of T cells expressing anti-CD20 CARs either in vitro or invivo (see, also, Gall et al., Exp. Hematol. 33:452, 2005). Moreover,mouse experiments of this disclosure demonstrate groups receiving acombination therapy had outcomes as good as or better than mice treatedwith CAR T cells alone.

Prior to setting forth this disclosure in more detail, it may be helpfulto an understanding thereof to provide definitions of certain terms tobe used herein. Additional definitions are set forth throughout thisdisclosure.

In the present description, any concentration range, percentage range,ratio range, or integer range is to be understood to include the valueof any integer within the recited range and, when appropriate, fractionsthereof (such as one tenth and one hundredth of an integer), unlessotherwise indicated. Also, any number range recited herein relating toany physical feature, such as polymer subunits, size or thickness, areto be understood to include any integer within the recited range, unlessotherwise indicated. As used herein, the term “about” means±20% of theindicated range, value, or structure, unless otherwise indicated. Itshould be understood that the terms “a” and “an” as used herein refer to“one or more” of the enumerated components. The use of the alternative(e.g., “or”) should be understood to mean either one, both, or anycombination thereof of the alternatives. As used herein, the terms“include,” “have” and “comprise” are used synonymously, which terms andvariants thereof are intended to be construed as non-limiting.

“Optional” or “optionally” means that the subsequently describedelement, component, event, or circumstance may or may not occur, andthat the description includes instances in which the element, component,event, or circumstance occurs and instances in which they do not.

In addition, it should be understood that the individual constructs, orgroups of constructs, derived from the various combinations of thestructures and subunits described herein, are disclosed by the presentapplication to the same extent as if each construct or group ofconstructs was set forth individually. Thus, selection of particularstructures or particular subunit is within the scope of the presentdisclosure.

The term “consisting essentially of” limits the scope of a claim to thespecified materials or steps, or to those that do not materially affectthe basic characteristics of a claimed invention. For example, a proteindomain, region, module, or fragment (e.g., a binding domain, hingeregion, linker module, or tag) or a protein (which may have one or moredomains, regions, or modules) “consists essentially of” a particularamino acid sequence when the amino acid sequence of a domain, region,module, fragment, or protein includes insertions, deletions,substitutions, or a combination thereof (e.g., addition of amino acidsat the amino- or carboxy-terminus, or between domains) that, incombination, contribute to at most 20% (e.g., at most 15%, 10%, 8%, 6%,5%, 4%, 3%, 2% or 1%) of the length of a domain, region, module,cassette or protein and do not substantially affect (i.e., do not reducethe activity by more than 50%, such as no more than 40%, 30%, 25%, 20%,15%, 10%, 5%, or 1%) the activity of the domain(s), region(s),module(s), cassette(s), or protein (e.g., the target binding affinity ofa binding domain).

As used herein, “amino acid” refers to naturally occurring and syntheticamino acids, as well as amino acid analogs and amino acid mimetics thatfunction in a manner similar to the naturally occurring amino acids.Naturally occurring amino acids are those encoded by the genetic code,as well as those amino acids that are later modified, e.g.,hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acidanalogs refer to compounds that have the same basic chemical structureas a naturally occurring amino acid, i.e., an α-carbon that is bound toa hydrogen, a carboxyl group, an amino group, and an R group, e.g.,homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) ormodified peptide backbones, but retain the same basic chemical structureas a naturally occurring amino acid. Amino acid mimetics refer tochemical compounds that have a structure that is different from thegeneral chemical structure of an amino acid, but that functions in amanner similar to a naturally occurring amino acid.

A “conservative substitution” refers to amino acid substitutions that donot significantly affect or alter binding characteristics of aparticular protein. Generally, conservative substitutions are ones inwhich a substituted amino acid residue is replaced with an amino acidresidue having a similar side chain. Conservative substitutions includea substitution found in one of the following groups: Group 1: Alanine(Ala or A), Glycine (Gly or G), Serine (Ser or S), Threonine (Thr or T);Group 2: Aspartic acid (Asp or D), Glutamic acid (Glu or Z); Group 3:Asparagine (Asn or N), Glutamine (Gln or Q); Group 4: Arginine (Arg orR), Lysine (Lys or K), Histidine (His or H); Group 5: Isoleucine (Ile orI), Leucine (Leu or L), Methionine (Met or M), Valine (Val or V); andGroup 6: Phenylalanine (Phe or F), Tyrosine (Tyr or Y), Tryptophan (Trpor W). Additionally or alternatively, amino acids can be grouped intoconservative substitution groups by similar function, chemicalstructure, or composition (e.g., acidic, basic, aliphatic, aromatic, orsulfur-containing). For example, an aliphatic grouping may include, forpurposes of substitution, Gly, Ala, Val, Leu, and Ile. Otherconservative substitutions groups include: sulfur-containing: Met andCysteine (Cys or C); acidic: Asp, Glu, Asn, and Gln; small aliphatic,nonpolar or slightly polar residues: Ala, Ser, Thr, Pro, and Gly; polar,negatively charged residues and their amides: Asp, Asn, Glu, and Gln;polar, positively charged residues: His, Arg, and Lys; large aliphatic,nonpolar residues: Met, Leu, Ile, Val, and Cys; and large aromaticresidues: Phe, Tyr, and Trp. Additional information can be found inCreighton (1984) Proteins, W.H. Freeman and Company.

As used herein, “protein” or “polypeptide refers to a polymer of aminoacid residues. Proteins apply to naturally occurring amino acidpolymers, as well as to amino acid polymers in which one or more aminoacid residue is an artificial chemical mimetic of a correspondingnaturally occurring amino acid and non-naturally occurring amino acidpolymers.

“Percent sequence identity” refers to a relationship between two or moresequences, as determined by comparing the sequences. Preferred methodsto determine sequence identity are designed to give the best matchbetween the sequences being compared. For example, the sequences arealigned for optimal comparison purposes (e.g., gaps can be introduced inone or both of a first and a second amino acid or nucleic acid sequencefor optimal alignment). Further, non-homologous sequences may bedisregarded for comparison purposes. The percent sequence identityreferenced herein is calculated over the length of the referencesequence, unless indicated otherwise. Methods to determine sequenceidentity and similarity can be found in publicly available computerprograms. Sequence alignments and percent identity calculations may beperformed using a BLAST program (e.g., BLAST 2.0, BLASTP, BLASTN, orBLASTX). The mathematical algorithm used in the BLAST programs can befound in Altschul et al., Nucleic Acids Res. 25:3389-3402, 1997. Withinthe context of this disclosure, it will be understood that wheresequence analysis software is used for analysis, the results of theanalysis are based on the “default values” of the program referenced.“Default values” mean any set of values or parameters which originallyload with the software when first initialized.

“Nucleic acid molecule” or “polynucleotide” refers to a polymericcompound including covalently linked nucleotides, which can be made upof natural subunits (e.g., purine or pyrimidine bases) or non-naturalsubunits (e.g., morpholine ring). Purine bases include adenine, guanine,hypoxanthine, and xanthine, and pyrimidine bases include uracil,thymine, and cytosine. Nucleic acid molecules include polyribonucleicacid (RNA), polydeoxyribonucleic acid (DNA), which includes cDNA,genomic DNA, and synthetic DNA, either of which may be single or doublestranded. If single stranded, the nucleic acid molecule may be thecoding strand or non-coding (anti-sense strand). A nucleic acid moleculeencoding an amino acid sequence includes all nucleotide sequences thatencode the same amino acid sequence. Some versions of the nucleotidesequences may also include intron(s) to the extent that the intron(s)would be removed through co- or post-transcriptional mechanisms. Inother words, different nucleotide sequences may encode the same aminoacid sequence as the result of the redundancy or degeneracy of thegenetic code, or by splicing.

Variants of nucleic acid molecules of this disclosure are alsocontemplated. Variant nucleic acid molecules are at least 70%, 75%, 80%,85%, 90%, and preferably 95%, 96%, 97%, 98%, 99%, or 99.9% identical anucleic acid molecule of a defined or reference polynucleotide asdescribed herein, or that hybridizes to a polynucleotide under stringenthybridization conditions of 0.015M sodium chloride, 0.0015M sodiumcitrate at about 65-68° C. or 0.015M sodium chloride, 0.0015M sodiumcitrate, and 50% formamide at about 42° C. Nucleic acid moleculevariants retain the capacity to encode a fusion protein or a bindingdomain thereof having a functionality described herein, such asspecifically binding a target molecule (e.g., CD20).

A “functional variant” refers to a polypeptide or polynucleotide that isstructurally similar or substantially structurally similar to a parentor reference compound of this disclosure, but differs slightly incomposition (e.g., one base, atom or functional group is different,added, or removed), such that the polypeptide or encoded polypeptide iscapable of performing at least one function of the encoded parentpolypeptide with at least 50% efficiency, preferably at least 55%, 60%,70%, 75%, 80%, 85%, 90%, 95% or 100% level of activity of the parentpolypeptide. In other words, a functional variant of a polypeptide orencoded polypeptide of this disclosure has “similar binding,” “similaraffinity” or “similar activity” when the functional variant displays nomore than a 50% reduction in performance in a selected assay as comparedto the parent or reference polypeptide, such as an assay for measuringbinding affinity (e.g., Biacore® or tetramer staining measuring anassociation (K_(a)) or a dissociation (K_(D)) constant).

As used herein, a “functional portion” or “functional fragment” refersto a polypeptide or polynucleotide that comprises only a domain, portionor fragment of a parent or reference compound, and the polypeptide orencoded polypeptide retains at least 50% activity associated with thedomain, portion or fragment of the parent or reference compound,preferably at least 55%, 60%, 70%, 75%, 80%, 85%, 90%, 95% or 100% levelof activity of the parent polypeptide, or provides a biological benefit(e.g., effector function). A “functional portion” or “functionalfragment” of a polypeptide or encoded polypeptide of this disclosure has“similar binding” or “similar activity” when the functional portion orfragment displays no more than a 50% reduction in performance in aselected assay as compared to the parent or reference polypeptide(preferably no more than 20% or 10%, or no more than a log difference ascompared to the parent or reference with regard to affinity), such as anassay for measuring binding affinity or measuring effector function(e.g., cytokine release).

As used herein, “heterologous” or “non-endogenous” or “exogenous” refersto any gene, protein, compound, nucleic acid molecule, or activity thatis not native to a host cell or a subject, or any gene, protein,compound, nucleic acid molecule, or activity native to a host cell or asubject that has been altered. Heterologous, non-endogenous, orexogenous includes genes, proteins, compounds, or nucleic acid moleculesthat have been mutated or otherwise altered such that the structure,activity, or both is different as between the native and altered genes,proteins, compounds, or nucleic acid molecules. In certain embodiments,heterologous, non-endogenous, or exogenous genes, proteins, or nucleicacid molecules (e.g., receptors, ligands, etc.) may not be endogenous toa host cell or a subject, but instead nucleic acids encoding such genes,proteins, or nucleic acid molecules may have been added to a host cellby conjugation, transformation, transfection, electroporation, or thelike, wherein the added nucleic acid molecule may integrate into a hostcell genome or can exist as extra-chromosomal genetic material (e.g., asa plasmid or other self-replicating vector). The term “homologous” or“homolog” refers to a gene, protein, compound, nucleic acid molecule, oractivity found in or derived from a host cell, species, or strain. Forexample, a heterologous or exogenous polynucleotide or gene encoding apolypeptide may be homologous to a native polynucleotide or gene andencode a homologous polypeptide or activity, but the polynucleotide orpolypeptide may have an altered structure, sequence, expression level,or any combination thereof. A non-endogenous polynucleotide or gene, aswell as the encoded polypeptide or activity, may be from the samespecies, a different species, or a combination thereof.

As used herein, the term “endogenous” or “native” refers to apolynucleotide, gene, protein, compound, molecule, or activity that isnormally present in a host cell or a subject.

“Expression” refers to transcription or translation of a nucleic acidmolecule that is operably linked to an expression control sequence(e.g., promoter).

As used herein, the term “engineered,” “recombinant” or “non-natural”refers to an organism, microorganism, cell, nucleic acid molecule, orvector that includes at least one genetic alteration or has beenmodified by introduction of an exogenous nucleic acid molecule, whereinsuch alterations or modifications are introduced by genetic engineering(i.e., human intervention). Genetic alterations include, for example,modifications introducing expressible nucleic acid molecules encodingproteins, fusion proteins or enzymes, or other nucleic acid moleculeadditions, deletions, substitutions or other functional disruption of acell's genetic material. Additional modifications include, for example,non-coding regulatory regions in which the modifications alterexpression of a polynucleotide, gene or operon.

As used herein, a “fusion protein” refers to a protein that, in a singlechain, has at least two distinct domains, wherein the domains are notnaturally found together in a protein. A polynucleotide encoding afusion protein may be constructed using PCR, recombinantly engineered,or the like, or such fusion proteins can be synthesized. A fusionprotein may further contain other components, such as a tag, a linkermodule or a transduction marker. In certain embodiments, a fusionprotein expressed or produced by a host cell (e.g., a T cell) locates toa cell surface, where the fusion protein is anchored to the cellmembrane (e.g., via a transmembrane domain) and comprises anextracellular portion (e.g., containing a binding domain) and anintracellular portion (e.g., containing a signaling domain, effectordomain, co-stimulatory domain or combinations thereof).

Terms understood by those in the art of antibody technology are eachgiven the meaning acquired in the art, unless expressly defineddifferently herein. The term “antibody” refers to an intact antibodycomprising at least two heavy (H) chains and two light (L) chainsinter-connected by disulfide bonds, as well as an antigen-bindingportion of an intact antibody that has or retains the capacity to bind atarget molecule. Antibodies include polyclonal and monoclonalantibodies. An antibody may be naturally occurring, recombinantlyproduced, genetically engineered, or modified, and includes modifiedforms of immunoglobulins, such as, for example intrabodies, peptibodies,nanobodies, single domain antibodies, multispecific antibodies (e.g.,bispecific antibodies, diabodies, triabodies, tetrabodies, tandemdi-scFV, tandem tri-scFv). “Antigen-binding portion,” “antigen-bindingfragment” or “antigen-binding domain” from an antibody refers to an“antibody fragment” that comprises a portion of an intact antibody andcontains the antigenic determining variable regions or complementarydetermining regions of an antibody. Examples of antibody fragmentsinclude Fab, Fab′, F(ab′)2, and Fv fragments, Fab′-SH, F(ab′)₂,diabodies, linear antibodies, single chain antibodies, scFv (i.e., afusion protein of the variable heavy (VH) and variable light (VL)regions of an Ig molecule, connected with a short linked peptide ofgenerally about 10 to about 25 amino acids), single domain antibodies(e.g., sdAb, sdFv, nanobody), and multispecific antibodies comprisingantibody fragments. A monoclonal antibody or antigen-binding portionthereof may be non-human, chimeric, humanized, or human, preferablyhumanized or human. Immunoglobulin structure and function are reviewed,for example, in Harlow et al., Eds., Antibodies: A Laboratory Manual,Chapter 14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, 1988). Anantibody may be of any class or subclass, including IgG and subclassesthereof (IgG₁, IgG₂, IgG₃, IgG₄), IgM, IgE, IgA, and IgD.

The terms “V_(L)” and “V_(H)” refer to the variable binding region froman antibody light and heavy chain, respectively. The variable bindingregions are made up of discrete, well-defined sub-regions known as“complementarity determining regions” (CDRs) and “framework regions”(FRs).

The terms “complementarity determining region” (CDR) or “hypervariableregion” (HVR) are known in the art to refer to non-contiguous sequencesof amino acids within antibody variable regions, which confer antigenspecificity or binding affinity. In general, there are three CDRs ineach heavy chain variable region (HCDR1, HCDR2, and HCDR3) and threeCDRs in each light chain variable region (LCDR1, LCDR2, and LCDR3).

“Framework regions” (FR) as used herein refer to the non-CDR portions ofthe variable regions of the heavy and light chains. In general, thereare four FRs in each full-length heavy chain variable region (FR-H1,FR-H2, FR-H3, and FR-H4), and four FRs in each full-length light chainvariable region (FR-L1, FR-L2, FR-L3, and FR-L4).

The term “CL” refers to an “immunoglobulin light chain constant region”or a “light chain constant region,” i.e., a constant region from anantibody light chain. The term “CH” refers to an “immunoglobulin heavychain constant region” or a “heavy chain constant region,” which isfurther divisible, depending on the antibody isotype into CH1, CH2, andCH3 (IgA, IgD, IgG), or CH1, CH2, CH3, and CH4 domains (IgE, IgM).

A “fragment antigen binding” (Fab) region is a part of an antibody thatbinds to antigens, and includes the variable region and CH1 of the heavychain linked to the light chain via an inter-chain disulfide bond. A“fragment crystallizable” (Fc) region is a part of an antibody that isnot a Fab region, and includes the CH regions other than CH1 (e.g., CH2and CH3 of an IgG, IgA, or IgD antibody, or CH2, CH3, and CH4 of an IgEantibody). By way of background, an Fc region is responsible for theeffector functions of an immunoglobulin, such as antibody-dependentcell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity(CDC) and complement fixation, binding to Fc receptors (e.g., CD16,CD32, FcRn), greater half-life in vivo relative to a polypeptide lackingan Fc region, protein A binding, and perhaps even placental transfer(see Capon et al., Nature 337:525, 1989).

As used herein, “Fc region portion” refers to the heavy chain constantregion segment of an Fc fragment from an antibody, which can include oneor more constant domains, such as CH2, CH3, CH4, or any combinationthereof. In certain embodiments, an Fc region portion includes the CH2and CH3 domains of an IgG, IgA, or IgD antibody or any combinationthereof, or the CH2, CH3, and CH4 domains of an IgM or IgE antibody andany combination thereof. In other embodiments, a CH2CH3 or a CH3CH4structure has sub-region domains from the same antibody isotype and arehuman, such as human IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgD, IgE, orIgM (e.g., CH2CH3 from human IgG1 or IgG4). In certain embodiments, anFc region portion found in fusion proteins of the present disclosurewill be capable of mediating one or more of effector functions of animmunoglobulin, will be capable of mediating one or more enhancedeffector functions, or will lack one or more or all of these activitiesby way of, for example, one or more mutations known in the art.

In addition, antibodies have a hinge sequence that is typically situatedbetween the Fab and Fc region (but a lower section of the hinge mayinclude an amino-terminal portion of the Fc region). By way ofbackground, an immunoglobulin hinge acts as a flexible spacer to allowthe Fab region to move freely in space. In contrast to the constantregions, hinges are structurally diverse, varying in both sequence andlength between immunoglobulin classes and even among subclasses. Forexample, a human IgG1 hinge region is freely flexible, which allows theFab regions to rotate about their axes of symmetry and move within asphere centered at the first of two inter-heavy chain disulfide bridges.By comparison, a human IgG2 hinge is relatively short and contains arigid poly-proline double helix stabilized by four inter-heavy chaindisulfide bridges, which restricts the flexibility. A human IgG3 hingediffers from the other subclasses by its unique extended hinge region(about four times as long as the IgG1 hinge), containing 62 amino acids(including 21 prolines and 11 cysteines), forming an inflexiblepoly-proline double helix and providing greater flexibility because theFab regions are relatively far away from the Fc region. A human IgG4hinge is shorter than IgG1 but has the same length as IgG2, and itsflexibility is intermediate between that of IgG1 and IgG2.

A “T cell” is an immune system cell that matures in the thymus andproduces T cell receptors (TCRs). T cells can be naïve (not exposed toantigen; increased expression of CD62L, CCR7, CD28, CD3, CD127, andCD45RA, and decreased expression of CD45RO as compared to T_(CM)),memory T cells (T_(M)) (antigen-experienced and long-lived), andeffector cells (antigen-experienced, cytotoxic). T_(M) can be furtherdivided into subsets of central memory T cells (T_(CM), increasedexpression of CD62L, CCR7, CD28, CD127, CD45RO, and CD95, and decreasedexpression of CD54RA as compared to naïve T cells) and effector memory Tcells (T_(EM), decreased expression of CD62L, CCR7, CD28, CD45RA, andincreased expression of CD127 as compared to naïve T cells or T_(CM)).Effector T cells (T_(E)) refers to antigen-experienced CD8⁺ cytotoxic Tlymphocytes that have decreased expression of CD62L, CCR7, CD28, and arepositive for granzyme and perforin as compared to T_(CM). T helper cells(T_(H)) release cytokines to aid in antigen signaling and, when mature,express the surface protein CD4 (are CD4⁺)As used herein, “T cells” or“T lymphocytes” are from any mammal, including primates, dogs, orhorses, preferably humans. In some embodiments, T cells are autologous,allogeneic, or syngeneic.

“T cell receptor” (TCR) refers to a molecule found on the surface of Tcells (or T lymphocytes) that, in association with CD3, is generallyresponsible for recognizing antigens bound to major histocompatibilitycomplex (MHC) molecules. The TCR has a disulfide-linked heterodimer ofthe highly variable α and β chains (also known as TCRα and TCRβ,respectively) in most T cells. In a small subset of T cells, the TCR ismade up of a heterodimer of variable γ and δ chains (also known as TCRγand TCRδ, respectively). Each chain of the TCR is a member of theimmunoglobulin superfamily and possesses one N-terminal immunoglobulinvariable domain, one immunoglobulin constant domain, a transmembraneregion, and a short cytoplasmic tail at the C-terminal end (see, Janewayet al., Immunobiology: The Immune System in Health and Disease, 3^(rd)Ed., Current Biology Publications, p. 4:33, 1997). TCR, as used in thepresent disclosure, may be from various animal species, including human,mouse, rat, cat, dog, goat, horse, or other mammals. TCRs may becell-bound (i.e., have a transmembrane region or domain) or in solubleform. As discussed herein, a binding domain according to the presentdisclosure may comprise a single-chain TCR (scTCR), which is analogousto an scFv derived from an immunoglobulin and comprises the variabledomains from TCRα and TCRβ chains linked together using, e.g., a peptideor non-peptide linker and optionally through disulfide bonding.

“Major histocompatibility complex molecules” (MHC molecules) refer toglycoproteins that deliver peptide antigens to a cell surface. MHC classI molecules are heterodimers consisting of a membrane spanning α chain(with three a domains) and a non-covalently associated β2 microglobulin.MHC class II molecules are composed of two transmembrane glycoproteins,α and β, both of which span the membrane. Each chain has two domains.MHC class I molecules deliver peptides originating in the cytosol to thecell surface, where a peptide:MHC complex is recognized by CD8⁺ T cells.MHC class II molecules deliver peptides originating in the vesicularsystem to the cell surface, where they are recognized by CD4⁺ T cells.An MHC molecule may be from various animal species, including human,mouse, rat, cat, dog, goat, horse, or other mammals.

“Cells of T cell lineage” refer to cells that show at least onephenotypic characteristic of a T cell, or a precursor or progenitorthereof that distinguishes the cells from other lymphoid cells, andcells of the erythroid or myeloid lineages. Such phenotypiccharacteristics can include expression of one or more proteins specificfor T cells (e.g., CD3⁺, CD4⁺, CD8⁺), or a physiological, morphological,functional, or immunological feature specific for a T cell. For example,cells of the T cell lineage may be progenitor or precursor cellscommitted to the T cell lineage; CD25⁺ immature and inactivated T cells;cells that have undergone CD4 or CD8 linage commitment; thymocyteprogenitor cells that are CD4⁺CD8⁺ double positive; single positive CD4⁺or CD8⁺; TCRαβ or TCR γδ; or mature and functional or activated T cells.

As used herein, “enriched” or “depleted” with respect to amounts of celltypes in a mixture refers to an increase in the number of the “enriched”type, a decrease in the number of the “depleted” cells, or both, in amixture of cells resulting from one or more enriching or depletingprocesses or steps. Thus, depending upon the source of an originalpopulation of cells subjected to an enriching process, a mixture orcomposition may contain 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% ormore (in number or count) of the “enriched” cells. Cells subjected to adepleting process can result in a mixture or composition containing 50%,45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%,or 1% percent or less (in number or count) of the “depleted” cells. Incertain embodiments, amounts of a certain cell type in a mixture will beenriched and amounts of a different cell type will be depleted, such asenriching for CD4⁺ cells while depleting CD8⁺ cells, or enriching forCD62L⁺ cells while depleting CD62L⁻ cells, or combinations thereof.

“Treat” or “treatment” or “ameliorate” refers to medical management of adisease, disorder, or condition of a subject (e.g., a human or non-humanmammal, such as a primate, horse, cat, dog, goat, mouse, or rat). Ingeneral, an appropriate dose or treatment regimen comprising a host cellexpressing a fusion protein, the fusion protein comprising anextracellular component and an intracellular component connected by ahydrophobic portion, wherein the extracellular component comprises abinding domain that specifically binds CD20 and the intracellularcomponent comprises an effector domain of this disclosure, andoptionally an adjuvant, is administered in an amount sufficient toelicit a therapeutic or prophylactic benefit. Therapeutic orprophylactic/preventive benefit includes improved clinical outcome;lessening or alleviation of symptoms associated with a disease;decreased occurrence of symptoms; improved quality of life; longerdisease-free status; diminishment of extent of disease, stabilization ofdisease state; delay of disease progression; remission; survival;prolonged survival; or any combination thereof. As further describedherein, a treatment regimen may comprise a combination therapy in whichone or more CD20-specific binding molecules, such as, for example,anti-CD20 antibodies are administered prior to, simultaneous with,contemporaneous with, or subsequent to administration of one or moresecond or adjunctive therapeutic. Exemplary anti-CD20 antibodiessuitable for use in the therapeutic methods described herein include1.5.3, 1F5, Leu16, rituximab, ofatumumab, veltuzumab, ublituximab, andocrelizumab.

A “therapeutically effective amount” or “effective amount” of aCD20-specific binding molecule, a fusion protein, or host cellexpressing a fusion protein of this disclosure (e.g., CD20 CAR) refersto an amount of CD20-specific binding molecules, fusion proteins, orhost cells sufficient to result in a therapeutic effect, includingimproved clinical outcome; lessening or alleviation of symptomsassociated with a disease; decreased occurrence of symptoms; improvedquality of life; longer disease-free status; diminishment of extent ofdisease, stabilization of disease state; delay of disease progression;remission; survival; or prolonged survival in a statisticallysignificant manner. When referring to an individual active ingredient ora cell expressing a single active ingredient, administered alone, atherapeutically effective amount refers to the effects of thatingredient or cell expressing that ingredient alone. When referring to acombination, a therapeutically effective amount refers to the combinedamounts of active ingredients or combined adjunctive active ingredientwith a cell expressing an active ingredient that results in atherapeutic effect, whether administered serially or simultaneously. Acombination may also be a cell expressing more than one activeingredient, such as two different CD20 CARs, or one CD20 CAR and CD20TCR, or CD20 CAR and another relevant therapeutic.

The term “pharmaceutically acceptable excipient or carrier” or“physiologically acceptable excipient or carrier” refer to biologicallycompatible vehicles, e.g., physiological saline, which are described ingreater detail herein, that are suitable for administration to a humanor other non-human mammalian subject and generally recognized as safe ornot causing a serious adverse event.

As used herein, “statistically significant” refers to a p value of 0.050or less when calculated using the Students t-test and indicates that itis unlikely that a particular event or result being measured has arisenby chance.

As used herein, the term “adoptive immune therapy” or “adoptiveimmunotherapy” refers to administration of naturally occurring orgenetically engineered, disease antigen-specific immune cells (e.g., Tcells). Adoptive cellular immunotherapy may be autologous (immune cellsare from the recipient), allogeneic (immune cells are from a donor ofthe same species) or syngeneic (immune cells are from a donorgenetically identical to the recipient).

Fusion Proteins

In certain aspects, the present disclosure provides fusion proteinscomprising an extracellular component and an intracellular componentconnected by a hydrophobic portion.

An “extracellular component” comprises a binding domain thatspecifically binds CD20. A “binding domain” (also referred to as a“binding region” or “binding moiety”), as used herein, refers to amolecule, such as a peptide, oligopeptide, polypeptide, or protein thatpossesses the ability to specifically and non-covalently associate,unite, or combine with a target molecule (e.g., CD20). A binding domainincludes any naturally occurring, synthetic, semi-synthetic, orrecombinantly produced binding partner for a biological molecule orother target of interest. In some embodiments, a binding domain is anantigen-binding domain, such as an antibody or TCR, or functionalbinding domain or antigen-binding fragment thereof.

In certain embodiments, a binding domain comprises a variable regionlinker (e.g., scFv). A “variable region linker” specifically refers to afive amino acid to about 35 amino acid sequence that connects a heavychain immunoglobulin variable region (VH) to a light chainimmunoglobulin variable region (VL), or connects TCR V_(α/β) and C_(α/β)chains (e.g., V_(α)-C_(α), V_(β)-C_(β), V_(α)-V_(β)) or connects eachV_(α)-C_(α), V_(β)-C_(β), or V_(α)-V_(β) pair to a hinge or hydrophobicdomain, which provides a spacer function and flexibility sufficient forinteraction of the two sub-binding domains so that the resulting singlechain polypeptide retains a specific binding affinity to the same targetmolecule as an antibody or TCR.

In certain embodiments, a variable region linker comprises from aboutten amino acids to about 30 amino acids or from about 15 amino acids toabout 25 amino acids. In particular embodiments, a variable regionlinker peptide comprises from one to ten repeats of Gly_(x)Ser_(y) (SEQID NO: 99), wherein x and y are independently an integer from 0 to 10,provided that x and y are not both 0 (e.g., Gly₄Ser (SEQ ID NO: 100)),Gly₃Ser (SEQ ID NO: 101), Gly₂Ser, or (Gly₃Ser)_(n)(Gly₄Ser)_(n) (SEQ IDNO: 102), (Gly₃Ser)_(n)(Gly₂Ser)_(n) (SEQ ID NO: 103),(Gly₃Ser)_(n)(Gly₄Ser)_(n) (SEQ ID NO: 104), or (Gly₄Ser)_(n) (SEQ IDNO: 105), wherein n is an integer of 1, 2, 3, 4, 5, or 6) and whereinlinked variable regions form a functional immunoglobulin-like bindingdomain (e.g., scFv or scTCR).

Exemplary binding domains include single chain antibody variable regions(e.g., domain antibodies, sFv, scFv, or Fab), antigen-binding regions ofTCRs, such as single chain TCRs (scTCRs), or synthetic polypeptidesselected for the specific ability to bind to a biological molecule.

As used herein, “specifically binds” refers to an association or unionof a binding domain, or a fusion protein thereof, to a target moleculewith an affinity or K_(a) (i.e., an equilibrium association constant ofa particular binding interaction with units of 1/M) equal to or greaterthan 10⁵M⁻¹, while not significantly associating or uniting with anyother molecules or components in a sample. Binding domains (or fusionproteins thereof) may be classified as “high affinity” binding domains(or fusion proteins thereof) or “low affinity” binding domains (orfusion proteins thereof). “High affinity” binding domains (or fusionproteins thereof) refer to those binding domains (or fusion proteinsthereof) with a K_(a) of at least 10⁷M⁻¹, at least 10⁸M⁻¹, at least10⁹M⁻¹, at least 10¹⁰ M⁻¹, at least 10¹¹M⁻¹, at least 10¹²M⁻¹, or atleast 10¹³ M⁻¹. “Low affinity” binding domains (or fusion proteinsthereof) refer to those binding domains (or fusion proteins thereof)with a K_(a) of up to 10⁷ M⁻¹, up to 10⁶ M⁻¹, up to 10⁵ M⁻¹.

Alternatively, affinity may be defined as an equilibrium dissociationconstant (K_(d)) of a particular binding interaction with units of M(e.g., 10⁻⁵ M to 10⁻¹³ M). In certain embodiments, a binding domain mayhave “enhanced affinity,” which refers to a selected or engineeredbinding domain with stronger binding to a target antigen than a wildtype (or parent) binding domain. For example, enhanced affinity may bedue to a K_(a) (equilibrium association constant) for the target antigenthat is greater than the wild type binding domain, due to a K_(d)(dissociation constant) for the target antigen that is less than that ofthe wild type binding domain, or due to an off-rate (K_(off)) for thetarget antigen that is less than that of the wild type binding domain. Avariety of assays are known for identifying binding domains of thepresent disclosure that specifically bind a particular target, as wellas determining binding domain or fusion protein affinities, such asWestern blot, ELISA, and Biacore® analysis (see also, e.g., Scatchard etal., Ann. N.Y. Acad. Sci. 51:660, 1949; and U.S. Pat. Nos. 5,283,173,5,468,614, or an equivalent).

Analysis or computer modeling of the primary and secondary amino acidstructure of a binding domain to analyze the tertiary structure of aprotein may aid in identifying specific amino acid residues that can besubstituted, added, or deleted without significantly altering thestructure and as a consequence, potentially significantly reducing thebinding specificity and affinity of a binding domain.

In certain embodiments, a binding domain comprises a V_(H) region. Forexample, a V_(H) region in a binding domain of the present disclosurecan be derived from or based on a V_(H) of a known monoclonal antibodyand may contain one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10)insertions, one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) deletions,one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acidsubstitutions (e.g., conservative amino acid substitutions ornon-conservative amino acid substitutions), or a combination of theabove-noted changes, when compared with the V_(H) of a known monoclonalantibody. An insertion, deletion, or substitution may be anywhere in theV_(H) region, including at the amino-terminus, carboxy-terminus, or bothends of the region, provided that each CDR comprises zero changes or atmost one, two, three or four changes from a CDR of the V_(H) region of aknown monoclonal antibody, and provided a binding domain containing themodified V_(H) region specifically binds its target with an affinitysimilar to the wild type binding domain.

In certain embodiments, a binding domain comprises a V_(L) region. Forexample, a V_(L) region in a binding domain of the present disclosure isderived from or based on a V_(L) of a known monoclonal antibody and maycontain one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) insertions,one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) deletions, one or more(e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acid substitutions (e.g.,conservative amino acid substitutions), or a combination of theabove-noted changes, when compared with the V_(L) of a known monoclonalantibody. An insertion, deletion, or substitution may be anywhere in theV_(L) region, including at the amino-terminus, carboxy-terminus, or bothends of the region, provided that each CDR comprises zero changes or atmost one, two, three or four changes from a CDR of the V_(L) region of aknown monoclonal antibody, and provided a binding domain containing themodified V_(L) region specifically binds its target with an affinitysimilar to the wild type binding domain.

In certain embodiments, a binding domain comprises an amino acidsequence that is at least 90%, at least 91%, at least 92%, at least 93%,at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%, at least 99.5%, or 100% identical to an amino acid sequenceof a light chain variable region (V_(L)); e.g., to a V_(L) from 1.5.3(SEQ ID NO.:1), 1F5 (SEQ ID NO.:3), Leu16 (SEQ ID NO.:2), rituximab,ofatumumab, veltuzumab, ublituximab, or ocrelizumab.

In further embodiments, a binding domain comprises an amino acidsequence that is at least 90%, at least 91%, at least 92%, at least 93%,at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%, at least 99.5%, or 100% identical to an amino acid sequenceof a heavy chain variable region (V_(H)); e.g., to a V_(H) from 1.5.3(SEQ ID NO.:4), 1F5 (SEQ ID NO.:6), Leu16 (SEQ ID NO.:5), rituximab,ofatumumab, veltuzumab, ublituximab, or ocrelizumab.

In still further embodiments, a binding domain comprises (a) an aminoacid sequence that is at least 90%, at least 91%, at least 92%, at least93%, at least 94%, at least 95%, at least 96%, at least 97%, at least98%, at least 99%, at least 99.5%, or 100% identical to an amino acidsequence of a V_(L); e.g., to a V_(L) from 1.5.3 (SEQ ID NO.:1), 1F5(SEQ ID NO.:3), Leu16 (SEQ ID NO.:2), rituximab, ofatumumab, veltuzumab,ublituximab, or ocrelizumab; and (b) an amino acid sequence that is atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, atleast 99.5%, or 100% identical to an amino acid sequence of a V_(H);e.g., to a V_(H) from 1.5.3 (SEQ ID NO.:4), 1F5 (SEQ ID NO.:6), Leu16(SEQ ID NO.:5), rituximab, ofatumumab, veltuzumab, ublituximab, orocrelizumab. In any of the aforementioned embodiments, each CDR of theV_(L), V_(H), or both comprises zero changes or at most one, two, three,four, five or six changes, as compared to a parent monoclonal antibodyor fragment or derivative thereof that specifically binds to CD20,provided that a binding domain containing the modified V_(L), V_(H), orboth region specifically binds CD20 with an affinity similar to the wildtype binding domain.

In certain embodiments, a binding domain comprises an amino acidsequence that is at least 90%, at least 91%, at least 92%, at least 93%,at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%, at least 99.5%, or 100% identical to an amino acid sequenceof a scFv, e.g., a scFv from an antibody of 1.5.3 (SEQ ID NO.:64), 1F5(SEQ ID NO.:66), Leu16 (SEQ ID NO.:65), rituximab, ofatumumab,veltuzumab, ublituximab, or ocrelizumab, wherein each CDR of the scFvcomprises zero changes or at most one, two, three, four, five or sixchanges, as compared to the corresponding CDR of a parent monoclonalantibody or fragment or derivative thereof that specifically binds toCD20, provided that scFv containing one or more modified CDRsspecifically binds CD20 with an affinity similar to the wild type scFvor corresponding antibody.

In certain embodiments, a binding domain is encoded by a polynucleotidethat is at least 60%, at least 70%, at least 75%, at least 80%, at least85%, at least 90%, at least 95%, or 100% identical to a polynucleotidesequence encoding a light chain variable region (V_(L)); e.g., to aV_(L)-encoding polynucleotide from 1.5.3 (SEQ ID NO.:70), 1F5 (SEQ IDNO.:72), Leu16 (SEQ ID NO.:71), rituximab, ofatumumab, veltuzumab,ublituximab, or ocrelizumab.

In further embodiments, a binding domain comprises a polynucleotide thatis at least 60%, at least 70%, at least 75%, at least 80%, at least 85%,at least 90%, at least 95%, or 100% identical to a polynucleotidesequence encoding a heavy chain variable region (V_(H)); e.g., to aV_(H)-encoding polynucleotide from 1.5.3 (SEQ ID NO.:73), 1F5 (SEQ IDNO.:75), Leu16 (SEQ ID NO.:74), rituximab, ofatumumab, veltuzumab,ublituximab, or ocrelizumab.

In still further embodiments, a binding domain comprises (a) apolynucleotide that is at least 60%, at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, at least 95%, or 100% identicalto a polynucleotide sequence encoding a V_(L); e.g., to a V_(L)-encodingpolynucleotide from 1.5.3 (SEQ ID NO.:70), 1F5 (SEQ ID NO.:72), Leu16(SEQ ID NO.:71), rituximab, ofatumumab, veltuzumab, ublituximab, orocrelizumab; and (b) a polynucleotide that is at least 60%, at least70%, at least 75%, at least 80%, at least 85%, at least 90%, at least95%, or 100% identical to a polynucleotide sequence encoding a V_(H);e.g., to a V_(H)-encoding polynucleotide from 1.5.3 (SEQ ID NO.:73), 1F5(SEQ ID NO.:75), Leu16 (SEQ ID NO.:74), rituximab, ofatumumab,veltuzumab, ublituximab, or ocrelizumab. In any of the aforementionedembodiments, polynucleotides encoding each CDR of the V_(L), V_(H), orboth comprises zero changes or at most one to six nucleotide changes, ascompared to a polynucleotide encoding a parent monoclonal antibody orfragment or derivative thereof that specifically binds to CD20, providedthat a binding domain containing the modified V_(L), V_(H), or bothregions specifically binds CD20 with an affinity similar to the wildtype binding domain.

In certain embodiments, a binding domain comprises a polynucleotide thatis at least 60%, at least 70%, at least 75%, at least 80%, at least 85%,at least 90%, at least 95%, or 100% identical to a polynucleotidesequence encoding a scFv, e.g., an encoded scFv comprising variabledomains from an antibody of 1.5.3 (SEQ ID NO.:67), 1F5 (SEQ ID NO.:69),Leu16 (SEQ ID NO.:68), rituximab, ofatumumab, veltuzumab, ublituximab,or ocrelizumab. In each of the aforementioned embodiments,polynucleotide sequences encoding each CDR of a scFv comprises zerochanges or at most one to six nucleotide changes, as compared to apolynucleotide encoding a parent scFv from a monoclonal antibody thatspecifically binds to CD20, provided that scFv containing one or moremodified CDRs specifically binds CD20 with an affinity similar to thewild type scFv or corresponding antibody.

In any of the embodiments described herein, a binding domain mayconsist, comprise, be based on or be derived from a V_(H), a V_(L), orboth, from ublituximab (see, e.g., US 2015/0290317), rituximab (see,e.g., US 2014/0004037), ocrelizumab (see, e.g., U.S. Pat. No.8,679,767), ofatumumab (see, e.g., US 2009/0169550), or veltuzumab (see,e.g., US 2009/0169550), the nucleotide and amino acid sequences of whichare herein incorporated by reference in their entirety. Additionally, inany of the methods described herein, a CD20 binding molecule maycomprise rituximab, ofatumumab, veltuzumab, or ocrelizumab, ublituximab,or any combination thereof.

A fusion protein of the present disclosure comprises an intracellularcomponent that comprises an effector domain. As used herein, an“effector domain” is an intracellular portion or domain of a fusionprotein or receptor that can directly or indirectly promote a biologicalor physiological response in a cell when receiving an appropriatesignal. In certain embodiments, an effector domain is from or a portionof a protein or protein complex that receives a signal when bound, orwhen the protein or portion thereof or protein complex binds directly toa target molecule, and triggers a signal from the effector domain. Aneffector domain may directly promote a cellular response when itcontains one or more signaling domains or motifs, such as animmunoreceptor tyrosine-based activation motif (ITAM), as found incostimulatory molecules. A costimulatory molecule or portion thereofcomprising ITAMs are generally known to be capable of initiating T cellactivation signaling following ligand engagement. In furtherembodiments, an effector domain will indirectly promote a cellularresponse by associating with one or more other proteins that directlypromote a cellular response.

In certain embodiments, an effector domain comprises a lymphocytereceptor signaling domain (e.g., CD3ζ), comprises a polypeptide havingone or more ITAMs from a costimulatory molecule (e.g., CD28, 4-1BB(CD137), OX40 (CD134)), or combinations thereof. In still furtherembodiments, an effector domain comprises a cytoplasmic portion thatassociates with a cytoplasmic signaling protein, wherein the cytoplasmicsignaling protein is a lymphocyte receptor or signaling domain thereof,a protein comprising a plurality of ITAMs, a costimulatory factor, orany combination thereof.

Exemplary effector domains include those from 4-1BB (CD137), CD3ε, CD3δ,CD3ζ, CD25, CD27, CD28, CD79A, CD79B, CARD11, DAP10, FcRα, FcRβ, FcRγ,Fyn, HVEM, ICOS, Lck, LAG3, LAT, LRP, NKG2D, NOTCH1, NOTCH2, NOTCH3,NOTCH4, Wnt, OX40 (CD134), ROR2, Ryk, SLAMF1, Slp76, pTα, TCRα, TCRβ,TRIM, Zap70, PTCH2, or any combination thereof.

In certain embodiments, an effector domain comprises a portion or domainfrom costimulatory molecule CD28, which may optionally include a LL→GGmutation at positions 186-187 of the native CD28 protein (SEQ ID NO.:15;see Nguyen et al., Blood 102:4320, 2003). In further embodiments, aneffector domain comprises CD3ζ or a functional portion thereof (SEQ IDNO.:17) and one or more portions or domains from a costimulatorymolecule, such as CD28 (SEQ ID NO.:15), 4-1BB (SEQ ID NO.:16), CD27, orOX40. In particular embodiments, an effector domain of a fusion proteinof the instant disclosure comprises an effector domains or a functionalportion thereof from CD3ζ (SEQ ID NO.:17) and CD28 (SEQ ID NO.:15); CD3ζ(SEQ ID NO.:17) and 4-1BB (SEQ ID NO.:16); or CD3ζ (SEQ ID NO.:17), CD28(SEQ ID NO.:15), and 4-1BB (SEQ ID NO.:16).

In certain embodiments, an effector domain comprises CD3ζ or afunctional portion thereof, which is encoded by a polynucleotide havingat least 60%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, or 100% sequence identity with SEQ ID NO.:86.In further embodiments, an effector domain comprises a portion or adomain from costimulatory molecule CD28, which is encoded by apolynucleotide having at least 60%, at least 70%, at least 75%, at least80%, at least 85%, at least 90%, at least 95%, or 100% sequence identityto SEQ ID NO.:84. In still further embodiments, an effector domaincomprises a portion or a domain from costimulatory molecule 4-1BB, whichis encoded by a polynucleotide having at least 60%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, or100% sequence identity to SEQ ID NO.:85.

An extracellular domain and an intracellular domain of the presentdisclosure are connected by a hydrophobic portion. A “hydrophobicportion,” as used herein, means any amino acid sequence having athree-dimensional structure that is thermodynamically stable in a cellmembrane, and generally ranges in length from about 15 amino acids toabout 30 amino acids. The structure of a hydrophobic portion maycomprise an alpha helix, a beta barrel, a beta sheet, a beta helix, orany combination thereof. In certain embodiments, a hydrophobic portionis comprised of a “transmembrane domain” from a known transmembraneprotein, which is a portion of the transmembrane protein that can insertinto or span a cell membrane. In some embodiments, a hydrophobic portionis a transmembrane domain, such as a CD4 transmembrane domain, CD8transmembrane domain, CD28 (e.g., SEQ ID NO.:14), CD27 transmembranedomain, and 4-1BB transmembrane domain. In certain embodiments, ahydrophobic portion is a CD28 transmembrane domain (SEQ ID NO.:14). Infurther embodiments, a hydrophobic portion is a CD28 transmembranedomain, which is encoded by a polynucleotide having at least 60%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 95%, or 100% sequence identity to SEQ ID NO.:83.

A fusion protein of the present disclosure may further comprise a linkermodule. A “linker module” may be an amino acid sequence having fromabout two amino acids to about 500 amino acids, which can provideflexibility and room for conformational movement between two regions,domains, motifs, fragments, or modules connected by a linker. In certainembodiments, a linker module may be located between a binding domain anda hydrophobic region. In such embodiments, a linker module can positiona binding domain away from the cell surface to enable proper cell/cellcontact, antigen binding, and activation (Patel et al., Gene Therapy 6:412-419, 1999). Linker module length may be varied to maximize tumorrecognition based on the selected target molecule, selected bindingepitope, or antigen binding domain size and affinity (see, e.g., Guestet al., J. Immunother. 28:203-11, 2005; PCT Publication No. WO2014/031687). Exemplary linker modules include those having aglycine-serine (Gly-Ser) linker having from one to about ten repeats ofGly_(x)Ser_(y), wherein x and y are independently an integer from 0 to10, provided that x and y are not both 0 (e.g., (Gly₄Ser)₂, (Gly₃Ser)₂,Gly₂Ser, or a combination thereof, such as (Gly₃Ser)₂Gly₂Ser). Incertain embodiments, a linker module comprises one or moreimmunoglobulin heavy chain constant regions, such as a CH3 alone, or aCH2CH3 structure, a CH3CH4 structure, an immunoglobulin hinge, or anycombination thereof (e.g., a CH2CH3 structure together with a hinge). Infurther embodiments, a linker module comprises all or a portion of an Fcdomain selected from: a CH1 domain, a CH2 domain, a CH3 domain, orcombinations thereof (see, e.g., PCT Publication WO 2014/031687).

Exemplary linker modules can vary in length, for instance, from aboutfive amino acids to about 500 amino acids, from about ten amino acids toabout 350 amino acids, from about 15 amino acids to about 100 aminoacids, from about 20 amino acids to about 75 amino acids, or from about25 amino acids to about 35 amino acids. In further embodiments, a linkermodule may further comprise a hinge region, a tag or both. Each suchcomponent of the linker module is not mutually exclusive.

In certain embodiments, a linker module of a fusion protein of thisdisclosure may include an IgG1 CH2 region with a N297Q mutation (SEQ IDNO.:10); an IgG4 CH2 region (SEQ ID NO.:11); an IgG1 CH3 region (SEQ IDNO.:12); or an IgG4 CH3 region (SEQ ID NO.:13). In certain embodiments,a linker module may include a glycine-serine linker (SEQ ID NO.:20,which may be encoded by SEQ ID NO.:89, or SEQ ID NO.:21, which may beencoded by SEQ ID NO.:90).

In further embodiments, a linker module of a fusion protein of thisdisclosure may include an IgG1 CH2 region with a N297Q mutation, whichis encoded by a polynucleotide having at least 60%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, or100% sequence identity with SEQ ID NO.:79. In other embodiments, alinker module of a fusion protein of this disclosure may include an IgG4CH2 region, which is encoded by a polynucleotide having at least 60%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 95%, or 100% sequence identity with SEQ ID NO.:80. In still otherembodiments, a linker module of a fusion protein of this disclosure mayinclude an IgG1 CH3 region, which is encoded by a polynucleotide havingat least 60%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, or 100% sequence identity with SEQ ID NO.:81.In yet other embodiments, a linker module of a fusion protein of thisdisclosure may include an IgG4 CH3 region, which is encoded by apolynucleotide having at least 60%, at least 70%, at least 75%, at least80%, at least 85%, at least 90%, at least 95%, or 100% sequence identitywith SEQ ID NO.:82.

In certain embodiments, a linker module further comprises a hingeregion. As used herein, a “hinge region” or a “hinge” refers to (a) animmunoglobulin hinge sequence (made up of, for example, upper and coreregions), or a functional fragment or variant thereof, (b) a type IIC-lectin interdomain (stalk) region, or a functional fragment or variantthereof, or (c) a cluster of differentiation (CD) molecule stalk region,or a functional variant thereof. As used herein, a “wild typeimmunoglobulin hinge region” refers to naturally occurring upper andmiddle hinge amino acid sequences interposed between and connecting theCH1 and CH2 domains found in the heavy chain of an antibody. In certainembodiments, a hinge region is human, and in particular embodiments,comprises a human IgG hinge region. In further embodiments, a hingeregion is an altered IgG4 hinge region as described in PCT PublicationNo. WO 2014/031687. In particular embodiments, a hinge region of afusion protein of this disclosure may be an IgG1 hinge (SEQ ID NO.:7).In related embodiments, a hinge region of a fusion protein of thisdisclosure may be an IgG1 hinge, which is encoded by a polynucleotide asset forth in SEQ ID NO.:76.

A fusion protein of the present disclosure may further comprise junctionamino acids. “Junction amino acids” or “junction amino acid residues”refer to one or more (e.g., about 2-20) amino acid residues between twoadjacent domains, motifs, regions, modules, or fragments of a protein,such as between a binding domain and an adjacent linker module, betweena hydrophobic domain and an adjacent effector domain, or on one or bothends of a linker module that links two domains, motifs, regions,modules, or fragments (e.g., between a linker module and an adjacentbinding domain or between a linker module and an adjacent hinge).Junction amino acids may result from the construct design of a fusionprotein (e.g., amino acid residues resulting from the use of arestriction enzyme site during the construction of a nucleic acidmolecule encoding a fusion protein). For example, a hydrophobic portionof a fusion protein may have one or more junction amino acids at theamino-terminal end, carboxy-terminal end, or both. Examples of junctionamino acids include junction amino acids from IgG2 (e.g., SEQ ID NO.:9,which may be encoded by SEQ ID NO.:78). In some embodiments where ahinge region is from IgG4, the hinge region can include junction aminoacids (e.g., SEQ ID NO.:8, which may be encoded by SEQ ID NO.:77). Incertain embodiments, a hydrophobic portion is a CD28 transmembranedomain having an amino acid of SEQ ID NO.:14 wherein the CD28transmembrane domain comprises an amino-terminal junction amino acid of,for example, methionine (see, e.g., fusion proteins of SEQ ID NO.:30,31, 39, and 40). Thus, in certain embodiments, a linker module comprisesan IgG4 hinge, IgG4 junction amino acids, and IgG4 CH2-CH3. In certainother embodiments, a linker module comprises an IgG1 hinge, IgG2junction amino acids, and IgG1 CH2-CH3.

In some embodiments, a fusion protein of the present disclosure mayfurther comprise a tag. As used herein, “tag” refers to a unique peptidesequence affixed to, fused to, or that is part of a protein of interest,to which a heterologous or non-endogenous cognate binding molecule(e.g., receptor, ligand, antibody, or other binding partner) is capableof specifically binding, where the binding property can be used todetect, identify, isolate or purify, track, enrich for, or target atagged protein or cells expressing a tagged protein, particularly when atagged protein is part of a heterogeneous population of proteins orother material, or when cells expressing a tagged protein are part of aheterogeneous population of cells (e.g., a biological sample likeperipheral blood). (See, e.g., WO 2015/095895.) In the provided fusionproteins, the ability of the tag(s) to be specifically bound by thecognate binding molecule(s) is distinct from, or in addition to, theability of the binding domain(s) to specifically bind to the targetmolecule(s). A tag generally is not an antigen-binding molecule, forexample, is not an antibody or TCR or an antigen-binding portionthereof. Examples of tags include Strep tag, His tag, Flag tag, Xpresstag, Avi tag, Calmodulin tag, Polyglutamate tag, HA tag, Myc tag, Nustag, S tag, X tag, SBP tag, Softag, V5 tag, CBP, GST, MBP, GFP,Thioredoxin tag. In particular embodiments, a Strep tag has an aminoacid sequence of SEQ ID NO.:62 or SEQ ID NO.:63.

A fusion protein of the present disclosure may comprise a signalpeptide. A “signal peptide” is a short (e.g., 5-30 amino acids) sequencethat is used to target the fusion protein for cell surface expression.Exemplary signal peptides include Granulocyte-macrophagecolony-stimulating factor (GM-CSF) signaling peptide (SEQ ID NO.:18,which may be encoded by SEQ ID NO.:87) and murine kappa signal peptide(SEQ ID NO.:19, which may be encoded by SEQ ID NO.:88).

In certain embodiments, a fusion protein is a chimeric antigen receptor.“Chimeric antigen receptor” (CAR) refers to a fusion protein of thepresent disclosure engineered to contain two or more naturally-occurringamino acid sequences linked together in a way that does not occurnaturally or does not occur naturally in a host cell, which fusionprotein can function as a receptor when present on a surface of a cell.

In some embodiments, a CAR is fully human or humanized. In certainembodiments, a CAR has a scFv from an anti-CD20 antibody or a scTCR froma TCR specific for a CD20 antigen. In particular embodiments, a CARcomprises a scFv from 1.5.3, 1F5, Leu16, rituximab, ofatumumab,veltuzumab, ocrelizumab, ublituximab, or any combination thereof. Inparticular embodiments, a CAR comprises a linker module comprising anIgG1 hinge, an IgG4 hinge, or any combination thereof. In furtherembodiments, a CAR comprises a linker module comprising an IgG1 CH2region with a N297Q mutation, an IgG4 CH2 region, an IgG1 CH3 region, anIgG4 CH3 region, or any combination thereof. In still furtherembodiments, a hydrophobic portion of a CAR comprises a CD28transmembrane domain. In some embodiments, a CAR comprises anintracellular domain comprising a portion or domain from CD3, 4-1BB,CD28, or any combination thereof. In any of the above embodiments, a CARcomprises junction amino acids between two adjacent domains, motifs,regions, modules, or fragments.

In certain embodiments, a CAR may be at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, at least 99.5%, or 100% identicalto 1.5.3-NQ-28-BB-z (SEQ ID NO.:26); 1.5.3-NQ-28-z (SEQ ID NO.:27);1.5.3-NQ-BB-z (SEQ ID NO.:28); 1.5.3-NQ-z (SEQ ID NO.:29); Leu16-28-BB-z(SEQ ID NO.:30); Leu16-28-z (SEQ ID NO.:31); 1F5-NQ-28-BB-z (SEQ IDNO.:32); 1F5-NQ-28-z (SEQ ID NO.:33); or 1F5-NQ-BB-z (SEQ ID NO.:34). Inparticular embodiments, a CAR comprise or consists of an amino acidsequence of any one of SEQ ID NOS.:26-34.

In certain embodiments, a CAR may be at least 60%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, or100% identical to a nucleic acid molecule sequence of any one of SEQ IDNOS.:44-52. In particular embodiments, a CAR is encoded by apolynucleotide comprising or consisting of a sequence of any one of SEQID NOS.:44-52.

Methods of making fusion proteins, including CARs, are well known in theart and are described, for example, in U.S. Pat. Nos. 6,410,319;7,446,191; U.S. Patent Publication No. 2010/065818; U.S. Pat. No.8,822,647; PCT Publication No. WO 2014/031687; U.S. Pat. No. 7,514,537;and Brentjens et al., 2007, Clin. Cancer Res. 13:5426.

Host Cells, Nucleic Acids and Vectors

In certain aspects, the present disclosure provides nucleic acidmolecules that encode any one or more of the fusion proteins describedherein. A polynucleotide encoding a desired fusion protein can beobtained or produced using recombinant methods known in the art usingstandard techniques, such as screening libraries from cells expressing adesired sequence or a portion thereof, by deriving a sequence from avector known to include the same, or by isolating a sequence or aportion thereof directly from cells or tissues containing the same.Alternatively, a sequence of interest can be produced synthetically.Such nucleic acid molecules can be inserted into an appropriate vector(e.g., viral vector or non-viral plasmid vector) for introduction into ahost cell of interest (e.g., an immune cell, such as a T cell).

A “vector” is a nucleic acid molecule that is capable of transportinganother nucleic acid. In some embodiments, vectors contain transcriptionor translation terminators, initiation sequences, or promoters forregulation of expression of a desired nucleic acid sequence. Vectors maybe, for example, plasmids, cosmids, viruses, or phage, or a transposonsystem (e.g., Sleeping Beauty, see, e.g., Geurts et al., Mol. Ther.8:108, 2003; Mátés et al., Nat. Genet. 41:753, 2009). An “expressionvector” is a vector that is capable of directing expression of a proteinencoded by one or more genes carried by a vector when it is present inthe appropriate environment.

A vector that encodes a core virus is referred to herein as a “viralvector.” There are a large number of available viral vectors suitablefor use with compositions of the instant disclosure, including thoseidentified for human gene therapy applications (see Pfeifer and Verma,Ann. Rev. Genomics Hum. Genet. 2:177, 2001). Suitable viral vectorsinclude vectors based on RNA viruses, such as retrovirus-derivedvectors.

“Retroviruses” are viruses having an RNA genome, which isreverse-transcribed into DNA using a reverse transcriptase enzyme, thereverse-transcribed DNA is then incorporated into the host cell genome.“Gammaretrovirus” refers to a genus of the retroviridae family. Examplesof gammaretroviruses include mouse stem cell virus, murine leukemiavirus, feline leukemia virus, feline sarcoma virus, and avianreticuloendotheliosis viruses. “Lentivirus” refers to another genus ofretroviruses that are capable of infecting dividing and non-dividingcells. Several examples of lentiviruses include human immunodeficiencyvirus (HIV; including HIV type 1, and HIV type 2); equine infectiousanemia virus; feline immunodeficiency virus (M); bovine immunedeficiency virus (BIV); and simian immunodeficiency virus (SIV).

In certain embodiments, the viral vector can be a gammaretrovirus, e.g.,Moloney murine leukemia virus (MLV)-derived vectors. In otherembodiments, the viral vector can be a more complex retrovirus-derivedvector, e.g., a lentivirus-derived vector. HIV-1-derived vectors belongto this category. Other examples include lentivirus vectors derived fromHIV-2, FIV, equine infectious anemia virus, SIV, and Maedi-Visna virus(ovine lentivirus). Methods of using retroviral and lentiviral viralvectors and packaging cells for transducing mammalian host cells withviral particles containing CAR transgenes are known in the art and havebeen previous described, for example, in U.S. Pat. No. 8,119,772;Walchli et al., PLoS One 6:327930, 2011; Zhao et al., J. Immunol.174:4415, 2005; Engels et al., Hum. Gene Ther. 14:1155, 2003; Frecha etal., Mol. Ther. 18:1748, 2010; Verhoeyen et al., Methods Mot Biol.506:97, 2009. Retroviral and lentiviral vector constructs and expressionsystems are also commercially available. Other viral vectors also can beused for polynucleotide delivery including DNA viral vectors, including,for example adenovirus-based vectors and adeno-associated virus(AAV)-based vectors; vectors derived from herpes simplex viruses (HSVs),including amplicon vectors, replication-defective HSV and attenuated HSV(Krisky et al., Gene Ther. 5: 1517, 1998).

Other vectors recently developed for gene therapy uses can also be usedwith the compositions and methods of this disclosure. Such vectorsinclude those derived from baculoviruses and α-viruses. (Jolly, D J.1999. Emerging Viral Vectors. pp 209-40 in Friedmann T. ed. TheDevelopment of Human Gene Therapy. New York: Cold Spring Harbor Lab), orplasmid vectors (such as sleeping beauty or other transposon vectors).

In certain embodiments, a viral vector is used to introduce anon-endogenous polynucleotide encoding a fusion protein specific for atarget, such as CD20. In such embodiments, a viral vector may be aretroviral vector or a lentiviral vector. A viral vector may alsoinclude nucleic acid sequences encoding a marker for transduction.Transduction markers for viral vectors are known in the art and includeselection markers, which may confer drug resistance, detectable markers,such as fluorescent markers or cell surface proteins that can bedetected by methods such as flow cytometry.

In certain embodiments, a viral vector comprises a transduction marker.As used herein, a “transduction marker” can be included in any of theconstructs as a way to monitor transfection efficiency or to detectcells expressing a fusion protein of interest. Exemplary transductionmarkers green fluorescent protein, an extracellular domain of human CD2,a truncated human EGFR (huEGFRt; SEQ ID NO.:25, which may be encoded bySEQ ID NO.:94; see Wang et al., Blood 118:1255, 2011), or a truncatedCD19 (SEQ ID NO.:24, which may be encoded by SEQ ID NO.:93). In certainembodiments, a viral vector comprises a suicide gene, such as iCasp9(see, e.g., Gargett and Brown, Front. Pharmacol. 5:235, 2104), or HSV-TK(see, e.g., Fillat et al., Curr. Gene Ther. 3:13, 2003).

When a viral vector genome comprises a plurality of polynucleotides tobe expressed in a host cell as separate transcripts, the viral vectormay also comprise additional sequences between the two (or more)transcripts allowing for bicistronic or multicistronic expression.Examples of such sequences used in viral vectors include internalribosome entry sites (IRES), furin cleavage sites, viral 2A peptide, orany combination thereof. In certain embodiments, a vector construct maycomprise a polynucleotide encoding a self-cleaving peptide (e.g., E2A(SEQ ID NO.:22, which may be encoded by SEQ ID NO.:91), T2A (SEQ IDNO.:23, which may be encoded by SEQ ID NO.:92), P2A (SEQ ID NO.:95,which may be encoded by SEQ ID NO.:97), or F2A (SEQ ID NO.:96, which maybe encoded by SEQ ID NO.:98)) such that the mature fusion protein doesnot contain a transduction marker or a suicide gene. In certainembodiments, a nucleic acid vector may encode a fusion peptide of thepresent disclosure, optionally containing a transduction marker (such astCD19 or tEGFR). In further embodiments, nucleic acid molecules encodinga fusion protein of this disclosure may be codon optimized to enhance ormaximize expression in certain types of cells, such as T cells (Scholtenet al., Clin. Immunol. 119: 135-145, 2006), and may optionally contain atransduction marker (such as tCD19 or tEGFR).

In any of the embodiments described herein, a vector containing apolynucleotide encoding a fusion protein of this disclosure may alsocontain a polynucleotide encoding a transduction marker, which may beused to target a host cell expressing the transduction marker forablation or death. It has been shown that the persistence of functionalantigen-targeting CAR T cells may cause sustained depletion of healthycells that endogenously express the antigen (see, e.g., Paskiewicz etal., J. Clin. Invest., 126(11):4262-4272 (2016). Thus, controlmechanisms that permit regulation (e.g., ablation, killing, or producinganother cytotoxic effect) of the transferred T cells after a achieving adesired antitumor affect are desirable. As used herein, the term“cytotoxic effect” encompasses ablating, killing, or otherwise impairingor reducing the ability of a cell to grow, divide, or survive.Non-limiting examples of cytotoxic effects include necrosis, lysis,apoptosis, swelling, loss of membrane integrity, reduced levels or ratesof transcription, reduced levels or rates of translation, reduced levelsor rates of ATP production, increased levels or rates of reactive oxygenspecies, reduced mitochondrial function, nuclear condensation, increasedcleavage of the cell's DNA, reduced rates of division or proliferation,and reduction or loss of specific cell function (e.g., the ability of aB lymphocyte to produce immunoglobulins). One exemplary approach is touse a marker (e.g., tEGFR) recognizable by an antibody (e.g., cetuximab)or antibody-drug conjugate that, upon binding the marker, facilitatesantibody-dependent cell-mediated cytotoxic (ADCC) orcomplement-dependent cytotoxic (CDC) responses, or delivers a cytotoxicmolecule, to ablate, kill, or otherwise cause a cytotoxic effect on thetransferred T cells. Thus, in certain embodiments, a vector comprises apolynucleotide encoding a fusion protein and comprises a polynucleotideencoding a transduction marker. A transduction marker that can bespecifically bound by a cytotoxic antibody, antibody-drug conjugate orother cytotoxic agent is referred to herein as “a suicide transductionmarker.” In certain embodiments, a method of treating a disease ordisorder associated with CD20 expression comprises administering atherapeutically effective amount of a transformed host cell to a subjectaccording to the present disclosure, wherein the transformed host cellcomprises a heterologous polynucleotide encoding a fusion protein and aheterologous polynucleotide encoding a suicide transduction marker,wherein the method optionally comprises administering a cytotoxicantibody, antibody-drug conjugate or other cytotoxic agent thatspecifically associates with, binds to or forms a complex with thesuicide transduction marker. In some embodiments, a suicide transductionmarker comprises or consists of a truncated EGFR (e.g., SEQ ID NO.: 25),which is specifically bound by an anti-EGFR antibody, such as, forexample, cetuximab. In further embodiments, a suicide transductionmarker comprises or consists of a truncated CD19 (e.g., SEQ ID NO.: 24),which is specifically bound by a cytotoxic anti-CD19 antibody orantibody-drug conjugate, such as, for example, blinatumomab,coltuximabravtansine, MOR208, MEDI-551, denintuzumabmafodotin, Merckpatent anti-CD19, taplutumomabpaptox, XmAb 5871, MDX-1342, SAR3419,SGN-19A, or AFM11 (see, e.g., Naddafi and Davami, Int. J. Mol. Cell.Med., 4(3):143-151 (2015)).

In any of the embodiments described herein, an encoded fusion protein ofthis disclosure may be a CAR, such as a CD20 specific CAR. In certainembodiments, a CAR or binding domain thereof encoded by a polynucleotidecontained in a vector of this disclosure is fully human or humanized. Infurther embodiments, a CAR encoded by a vector of this disclosure has ascFv from an anti-CD20 antibody or a scTCR from a TCR specific for aCD20 antigen. In still further embodiments, a CAR encoded by a vector ofthis disclosure comprises a scFv from 1.5.3, 1F5, Leu16, rituximab,ofatumumab, veltuzumab, ocrelizumab, ublituximab, or any combinationthereof. In particular embodiments, a CAR encoded by a polynucleotidecontained in a vector of this disclosure comprises a linker modulecomprising an IgG1 hinge, an IgG4 hinge, or any combination thereof. Infurther embodiments, a CAR encoded by a polynucleotide contained in avector of this disclosure comprises a linker module comprising an IgG1CH2 region with a N297Q mutation, an IgG4 CH2 region, an IgG1 CH3region, an IgG4 CH3 region, or any combination thereof. In particularembodiments, a linker module or a variable region linker of a CARencoded by a vector of this disclosure comprises a glycine-serinelinker. In still further embodiments, a hydrophobic portion of a CARencoded by a polynucleotide contained in a vector of this disclosurecomprises a CD28 transmembrane domain. In some embodiments, a CARencoded by a polynucleotide contained in a vector of this disclosurecomprises an intracellular domain comprising a portion or domain fromCD3ζ, 4-1BB, CD28, or any combination thereof. In any of the embodimentsdescribed herein, a CAR encoded by a polynucleotide contained in avector of this disclosure comprises junction amino acids betweenadjacent domains, motifs, regions, modules, or fragments.

In any of the embodiments described herein, a vector may comprise apolynucleotide that encodes a CAR that is at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, at least 99.5%, or 100% identicalto 1.5.3-NQ-28-BB-z (SEQ ID NO.:26); 1.5.3-NQ-28-z (SEQ ID NO.:27);1.5.3-NQ-BB-z (SEQ ID NO.:28); 1.5.3-NQ-z (SEQ ID NO.:29); Leu16-28-BB-z(SEQ ID NO.:30); Leu16-28-z (SEQ ID NO.:31); 1F5-NQ-28-BB-z (SEQ IDNO.:32); 1F5-NQ-28-z (SEQ ID NO.:33); or 1F5-NQ-BB-z (SEQ ID NO.:34). Infurther embodiments, a vector may comprise a polynucleotide that encodesa CAR that is comprised of or consists of an amino acid sequence of anyone of SEQ ID NOS.:26-34.

In still further embodiments, a CD20-specific CAR is encoded by apolynucleotide contained in a vector, wherein the polynucleotide has atleast 60%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, or 100% identity to a nucleic acid moleculesequence of any one of SEQ ID NOS.:44-52. In related embodiments, aCD20-specific CAR is encoded by a polynucleotide contained in a vector,wherein the polynucleotide comprises or consists of a sequence of anyone of SEQ ID NOS.:44-52.

Optionally, any vector of this disclosure containing a polynucleotidethat encodes a CAR of this disclosure can also encode a transductionmarker (e.g., tCD19), which may also include a self-cleaving peptide sothat the transduction marker and CAR are separated into separatemolecules—a CAR and a transduction marker. In certain embodiments, avector may comprise a polynucleotide encodes a self-cleaving peptidedisposed between a CD20-specific CAR and a tCD19 transduction marker,which polynucleotide is at least 60%, at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, at least 95%, or 100% identicalto a nucleic acid molecule sequence of any one of SEQ ID NOS.:53-61. Infurther embodiments, a vector may comprise a polynucleotide encoding aself-cleaving peptide disposed between a CD20-specific CAR and a tCD19transduction marker and that comprises or consists of a nucleic acidmolecule sequence of any one of SEQ ID NOS.:53-61.

In any of the embodiments disclosed herein, an isolated polynucleotideencodes a fusion protein capable of specifically binding CD20, whereinthe polynucleotide: (a) is at least 80% identical to a polynucleotidesequence of any one of SEQ ID NOS.:53-56; (b) is at least 80% identicalto a polynucleotide sequence of any one of SEQ ID NOS.:44-47; (c)comprises a polynucleotide sequence of any one of SEQ ID NOS.:53-56; (d)comprises a polynucleotide sequence of any one of SEQ ID NOS.:44-47; (e)consists of a polynucleotide sequence of any one of SEQ ID NOS.:53-56;or (f) consists of a polynucleotide sequence of any one of SEQ IDNOS.:44-47.

In any of the embodiments disclosed herein, a fusion protein is encodedby an isolated polynucleotide as disclosed herein. In certainembodiments, the fusion protein consists of or comprises an amino acidsequence wherein the fusion protein: (a) is at least 90% identical to amature fusion protein, wherein the mature fusion protein comprises anamino acid sequence of any one of SEQ ID NOS.:26-29 and 35-38 and 32-34with the tCD19 transduction marker removed; (b) is comprised of a maturefusion protein, wherein the mature fusion protein comprises an aminoacid sequence of any one of SEQ ID NOS.: 35-38 with the tCD19transduction marker removed; (c) consists of a mature fusion protein,wherein the mature fusion protein comprises an amino acid sequence ofany one of SEQ ID NOS.: 35-38 with the tCD19 transduction markerremoved; (d) is at least 90% identical to an amino acid sequence of anyone of SEQ ID NOS.:26 29; (e) is comprised of an amino acid sequence ofany one of SEQ ID NOS.:26 29; (f) consists of an amino acid sequence ofany one of SEQ ID NOS.:26 29.

In certain embodiments, a host cell is provided that comprises aheterologous polynucleotide as disclosed herein and is capable ofexpressing the fusion protein encoded by the heterologouspolynucleotide.

In any of the embodiments disclosed herein, a host cell comprises anisolated polynucleotide encoding a fusion protein capable ofspecifically binding CD20, wherein the polynucleotide: (a) is at least80% identical to a polynucleotide sequence of any one of SEQ IDNOS.:53-56; (b) is at least 80% identical to a polynucleotide sequenceof any one of SEQ ID NOS.:44-47; (c) comprises a polynucleotide sequenceof any one of SEQ ID NOS.:53-56; (d) comprises a polynucleotide sequenceof any one of SEQ ID NOS.:44-47; (e) consists of a polynucleotidesequence of any one of SEQ ID NOS.:53-56; or (f) consists of apolynucleotide sequence of any one of SEQ ID NOS.:44-47.

In certain embodiments, a host cell comprises a fusion protein thatconsists of or comprises an amino acid sequence wherein the fusionprotein: (a) is at least 90% identical to a mature fusion protein,wherein the mature fusion protein comprises an amino acid sequence ofany one of SEQ ID NOS.:26-29 and 35-38 and 32-34 with the tCD19transduction marker removed; (b) is comprised of a mature fusionprotein, wherein the mature fusion protein comprises an amino acidsequence of any one of SEQ ID NOS.: 35-38 with the tCD19 transductionmarker removed; (c) consists of a mature fusion protein, wherein themature fusion protein comprises an amino acid sequence of any one of SEQID NOS.: 35-38 with the tCD19 transduction marker removed; (d) is atleast 90% identical to an amino acid sequence of any one of SEQ IDNOS.:26 29; (e) is comprised of an amino acid sequence of any one of SEQID NOS.:26 29; (f) consists of an amino acid sequence of any one of SEQID NOS.:26 29.

In certain embodiments, a host cell comprises a heterologouspolynucleotide as disclosed herein and is capable of expressing thefusion protein encoded by the heterologous polynucleotide.

In certain embodiments, a host cell comprises a heterologouspolynucleotide encoding a fusion protein comprising a binding domain,wherein the binding domain is: (a) a 1.5.3 scFv comprising an amino acidsequence that is at least 90% identical to an amino acid sequence of SEQID NO.:64, wherein each CDR of the scFv comprises zero changes or atmost one, two, three, four, five or six changes as compared to thecorresponding CDR of a parent monoclonal antibody or fragment orderivative thereof that specifically binds to CD20, provided that scFvcontaining one or more modified CDRs specifically binds CD20 with anaffinity similar to the wild type scFv or corresponding antibody; (b) a1.5.3 scFv comprising or consisting of an amino acid sequence of SEQ IDNO.:64; (c) a 1F5 scFv comprising an amino acid sequence that is atleast 90% identical to an amino acid sequence of SEQ ID NO.:66, whereineach CDR of the scFv comprises zero changes or at most one, two, three,four, five or six changes as compared to the corresponding CDR of aparent monoclonal antibody or fragment or derivative thereof thatspecifically binds to CD20, provided that scFv containing one or moremodified CDRs specifically binds CD20 with an affinity similar to thewild type scFv or corresponding antibody; (d) a 1F5 scFv comprising orconsisting of an amino acid sequence of SEQ ID NO.:66; (e) a Leu16 scFvcomprising an amino acid sequence that is at least 90% identical to anamino acid sequence of SEQ ID NO.:65, wherein each CDR of the scFvcomprises zero changes or at most one, two, three, four, five or sixchanges as compared to the corresponding CDR of a parent monoclonalantibody or fragment or derivative thereof that specifically binds toCD20, provided that scFv containing one or more modified CDRsspecifically binds CD20 with an affinity similar to the wild type scFvor corresponding antibody; or (f) a Leu16 scFv comprising or consistingof an amino acid sequence of SEQ ID NO.:65.

In certain embodiments, a host cell comprises a heterologouspolynucleotide encoding a fusion protein comprising an scFv, wherein thescFv is encoded by: (a) a polynucleotide having at least 80% identity toa nucleic acid molecule sequence of SEQ ID NO.:67, whereinpolynucleotide sequences encoding each CDR of a scFv comprises zerochanges or at most one to six nucleotide changes, as compared to apolynucleotide encoding a parent scFv from a monoclonal antibody thatspecifically binds to CD20, provided that scFv containing one or moremodified CDRs specifically binds CD20 with an affinity similar to thewild type scFv or corresponding antibody; (b) a polynucleotidecomprising or consisting of a nucleic acid molecule sequence of SEQ IDNO.:67; (c) a polynucleotide having at least 80% identity to a nucleicacid molecule sequence of SEQ ID NO.:69, wherein polynucleotidesequences encoding each CDR of a scFv comprises zero changes or at mostone to six nucleotide changes, as compared to a polynucleotide encodinga parent scFv from a monoclonal antibody that specifically binds toCD20, provided that scFv containing one or more modified CDRsspecifically binds CD20 with an affinity similar to the wild type scFvor corresponding antibody; (d) a polynucleotide comprising or consistingof a nucleic acid molecule sequence of SEQ ID NO.:69; (e) apolynucleotide having at least 80% identity to a nucleic acid moleculesequence of SEQ ID NO.:68, wherein polynucleotide sequences encodingeach CDR of a scFv comprises zero changes or at most one to sixnucleotide changes, as compared to a polynucleotide encoding a parentscFv from a monoclonal antibody that specifically binds to CD20,provided that scFv containing one or more modified CDRs specificallybinds CD20 with an affinity similar to the wild type scFv orcorresponding antibody; or (f) a polynucleotide comprising or consistingof a nucleic acid molecule sequence of SEQ ID NO.:68.

In certain embodiments, a host cell comprises a heterologouspolynucleotide encoding a fusion protein, wherein the fusion protein isa chimeric antigen receptor and comprises or consists of an amino acidsequence that is at least 90% identical to an amino acid sequence of anyone of SEQ ID NOS.:26-3443.

In certain embodiments, a host cell comprises a heterologouspolynucleotide encoding a fusion protein comprising a hydrophobicportion, wherein the hydrophobic portion is a transmembrane domain. Incertain embodiments, the hydrophobic portion is a CD4, CD8, CD28 or CD27transmembrane domain.

In certain embodiments, a host cell comprises a heterologouspolynucleotide encoding a fusion protein comprising an effector domainor functional portion thereof, wherein the effector domain or functionalportion thereof is a 4-1BB (CD137), CD3ε, CD3δ, CD3ζ, CD25, CD27, CD28,CD79A, CD79B, CARD11, DAP10, FcRα, FCRβ, FcRγ, Fyn, HVEM, ICOS, Lck,LAG3, LAT, LRP, NKG2D, NOTCH1, NOTCH2, NOTCH3, NOTCH4, OX40 (CD134),ROR2, Ryk, SLAMF1, Slp76, pTα, TCRα, TCRβ, TRIM, Zap70, PTCH2, or anycombination thereof.

In certain embodiments, a host cell comprises a heterologouspolynucleotide encoding a fusion protein comprising an intracellularcomponent, wherein the intracellular component comprises: (a) a CD3ζeffector domain or functional portion thereof, a CD28 costimulatorydomain or functional portion thereof and a 4-1BB (CD137) costimulatorydomain or portion thereof; (b) a CD3ζ effector domain or functionalportion thereof, a CD28 costimulatory domain or functional portionthereof and a OX40 (CD134) costimulatory domain or portion thereof; (c)a CD3ζ effector domain or functional portion thereof, a CD27costimulatory domain or functional portion thereof and a 4-1BB (CD137)costimulatory domain or portion thereof; (d) a CD3ζ effector domain orfunctional portion thereof, a CD27 costimulatory domain or functionalportion thereof and a OX40 (CD134) costimulatory domain or portionthereof; (e) a CD3ζ effector domain or functional portion thereof, aCD27 costimulatory domain or functional portion thereof and a CD28costimulatory domain or portion thereof; or (f) a CD3ζ effector domainor functional portion thereof, a 4-1BB (CD137) costimulatory domain orfunctional portion thereof and a OX40 (CD134) costimulatory domain orportion thereof.

In any of the embodiments described herein, a vector containing a fusionprotein of this disclosure is transduced into a host cell.“Transduction” refers to introduction of a nucleic acid molecule (e.g.,a vector encoding a fusion protein of the present disclosure) into ahost cell. After transduction, a host cell may carry a vectorextra-chromosomally or integrated into a chromosome. Integration into ahost cell genome or self-replicating vectors generally result ingenetically stable inheritance of a transformed vector. Any suitabletransduction method can be utilized. A vector can be transferred into ahost cell by physical, chemical, or biological means. A host cellcontaining a transformed nucleic acid molecule is referred to as“engineered,” “recombinant,” or “non-natural.”

In certain embodiments, a cell, such as a T cell, obtained from asubject may be converted into an engineered, non-natural, or recombinantcell (e.g., an engineered, non-natural, or recombinant T cell) byintroducing a nucleic acid molecule encoding a cell surface locatedfusion protein as described herein, where the cell expresses the fusionprotein.

In certain embodiments, a host cell transfected to express a fusionprotein of this disclosure is a functional T cell, such as avirus-specific T cell, a tumor antigen specific cytotoxic T cell, anaïve T cell, a memory stem T cell, a central or effector memory T cell,γδ T cells, or a CD4+ CD25+ regulatory T cell. In further embodiments, anucleic acid molecule encoding a fusion protein of this disclosure isintroduced into bulk CD8+ T cells, naïve CD8+ T cells, CD8+ T_(CM)cells, CD8+ T_(EM) cells, or any combination thereof. In still furtherembodiments, a nucleic acid molecule encoding a fusion protein of thisdisclosure is introduced into bulk CD4+ T cells, naïve CD4+ T cells,CD4+ T_(CM) cells, CD4+ T_(EM) cells, or any combination thereof. Inother embodiments, a nucleic acid molecule encoding a fusion protein ofthis disclosure is introduced into a population of T cells enriched fornaïve CD8+ T cells and CD8+ T_(CM) cells. In still other embodiments, anucleic acid molecule encoding a fusion protein of this disclosure isintroduced into a population of T cells enriched for naïve CD4+ T cellsand CD4+ T_(CM) cells. In any of the aforementioned embodiments, the Tcells further contain a nucleic acid molecule encoding an engineeredCD20-specific TCR, an engineered CD20-specific high affinity TCR, aCD20-specific CAR, or any combination thereof.

In certain embodiments, prior to expansion and genetic modification ofthe T cells with a fusion protein construct of this disclosure, a sourceof T cells is obtained from a subject (e.g., peripheral bloodmononuclear cells (PBMCs), bone marrow, lymph node tissue, cord blood,thymus tissue, tissue from a site of infection, ascites, pleuraleffusion, or spleen tissue), from which T cells are isolated usingmethods known in the art. Specific T cell subsets can be collected inaccordance with known techniques and enriched or depleted by knowntechniques, such as affinity binding to antibodies, flow cytometry, orimmunomagnetic selection. After enrichment or depletion steps andintroduction of a fusion protein, in vitro expansion of the desiredmodified T cells can be carried out in accordance with known techniques(including those described in U.S. Pat. No. 6,040,177), or variationsthereof that will be apparent to those skilled in the art.

For example, a desired T cell population or subpopulation may beexpanded by adding an initial T cell population to a culture medium invitro, and then adding feeder cells, such as non-dividing PBMCs to theculture medium, (e.g., such that the resulting population of cellscontains at least about 5, 10, 20, or 40 or more PBMC feeder cells foreach T cell in the initial population to be expanded); and incubatingthe culture (e.g. for a time sufficient to expand the numbers of Tcells). Non-dividing feeder cells can comprise gamma-irradiated PBMCfeeder cells. In some embodiments, PBMCs are irradiated with gamma raysin the range of about 3000 to 3600 rads. The order of addition of Tcells and feeder cells to the culture media can be reversed if desired.A culture can typically be incubated under conditions of temperature andthe like that are suitable for the growth of T cells. For the growth ofhuman T lymphocytes, for example, the temperature will generally be atleast about 25° C., preferably at least about 30° C., more preferablyabout 37° C.

Optionally, expansion methods may further comprise adding non-dividingEpstein-Barr Virus (EBV)-transformed lymphoblastoid cells (LCL) asfeeder cells. LCL can be irradiated with gamma rays in the range ofabout 6000 to 10,000 rads. The LCL feeder cells may be provided in anysuitable amount, such as a ratio of LCL feeder cells to initial Tlymphocytes of at least about 10:1.

After isolation of T lymphocytes, both CD8+ cytotoxic and CD4+ helper Tlymphocytes can be sorted into naïve, memory, and effector T cellsubpopulations before genetically modifying with a fusion protein andexpanding. In certain embodiments, T cells that are modified to expressfusion proteins of this disclosure are bulk T cells (e.g., bulk CD4+ Tcells or bulk CD8+ T cells), or are a subpopulation of T cells, such ascentral memory T cells (e.g., CD8+ central memory T cells) or acombination of central memory (T_(CM)) and naïve (T_(N)) T cells (e.g.,CD4+ T_(CM)+ T_(N) cells).

In any of the embodiments described herein, a host cell (e.g., T cell)comprises a vector that contains a polynucleotide that encodes a CARthat is at least 90%, at least 91%, at least 92%, at least 93%, at least94%, at least 95%, at least 96%, at least 97%, at least 98%, at least99%, at least 99.5%, or 100% identical to 1.5.3-NQ-28-BB-z (SEQ IDNO.:26); 1.5.3-NQ-28-z (SEQ ID NO.:27); 1.5.3-NQ-BB-z (SEQ ID NO.:28);1.5.3-NQ-z (SEQ ID NO.:29); Leu16-28-BB-z (SEQ ID NO.:30); Leu16-28-z(SEQ ID NO.:31); 1F5-NQ-28-BB-z (SEQ ID NO.:32); 1F5-NQ-28-z (SEQ IDNO.:33); or 1F5-NQ-BB-z (SEQ ID NO.:34). In further embodiments, a hostcell (e.g., T cell) comprises a vector that contains a polynucleotidethat encodes a CAR that is comprised of or consists of an amino acidsequence of any one of SEQ ID NOS.:26-34. In any of these embodiments,the host cell is a T cell, wherein the T cells bulk CD4+ T cells, bulkCD8+ T cells, CD4+ central memory T cells, CD8+ central memory T cellsor a combination of CD4+ central memory (T_(CM)) and CD4+ naïve (T_(N))T cells. The CAR-modified CD4+ T cells and CAR-modified CD8+ T cells canbe mixed in a ratio of 3:1 to 1:1 to 1:3 before administration to asubject, or can be administered to a subject separately at the same orsimilar ratios.

In still further embodiments, a host cell (e.g., T cell) comprises avector that contains a polynucleotide that encodes a CD20-specific CAR,wherein the polynucleotide has at least 60%, at least 70%, at least 75%,at least 80%, at least 85%, at least 90%, at least 95%, or 100% identityto a nucleic acid molecule sequence of any one of SEQ ID NOS.:44-52. Inrelated embodiments, a host cell (e.g., T cell) comprises a vector thatcontains a polynucleotide that encodes a CD20-specific CAR, wherein thepolynucleotide comprises or consists of a sequence of any one of SEQ IDNOS.:44-52. In any of these embodiments, the host cell is a T cell,wherein the T cells bulk CD4+ T cells, bulk CD8+ T cells, CD4+ centralmemory T cells, CD8+ central memory T cells or a combination of CD4+central memory (T_(CM)) and CD4+ naïve (T_(N)) T cells. The CAR-modifiedCD4+ T cells and CAR-modified CD8+ T cells can be mixed in a ratio of3:1 to 1:1 to 1:3 before administration to a subject, or can beadministered to a subject separately at the same or similar ratios.

Optionally, a host cell comprising any vector of this disclosure thatcontains a polynucleotide that encodes a CAR of this disclosure can alsoencode a transduction marker (e.g., tCD19), which may also include aself-cleaving peptide so that the transduction marker and CAR areseparated into separate molecules—a CAR and a transduction marker. Incertain embodiments, a host cell (e.g., T cell) comprises a vector thatcontains a polynucleotide encoding a self-cleaving peptide disposedbetween a CD20-specific CAR and a tCD19 transduction marker, whichpolynucleotide is at least 60%, at least 70%, at least 75%, at least80%, at least 85%, at least 90%, at least 95%, or 100% identical to anucleic acid molecule sequence of any one of SEQ ID NOS.:53-61. Infurther embodiments, a host cell (e.g., T cell) comprises a vector thatcontains a polynucleotide encoding a self-cleaving peptide disposedbetween a CD20-specific CAR and a tCD19 transduction marker andcomprises or consists of a nucleic acid molecule sequence of any one ofSEQ ID NOS.:53-61.

Whether a T cell or T cell population is positive for a particular cellsurface marker can be determined by flow cytometry using staining with aspecific antibody for the surface marker and an isotype matched controlantibody. A cell population being “negative” for a marker refers to theabsence of significant staining of the cell population with a specificantibody above an isotype control, and “positive” refers to uniformstaining of the cell population above the levels found on an isotypecontrol. In some embodiments, a decrease in expression of one or moremarkers refers to a loss of 1 log 10 in the MFI or a percentage decreaseof T cells that exhibit the marker of at least 20% of the cells, 25% ofthe cells, 30% of the cells, 35% of the cells, 40% of the cells, 45% ofthe cells, 50% of the cells, 55% of the cells, 60% of the cells, 65% ofthe cells, 70% of the cells, 75% of the cells, 80% of the cells, 85% ofthe cells, 90% of the cell, 95% of the cells, or 100% of the cells, orany percentage between 20% and 100% when compared to a reference T cellpopulation. In some embodiments, a T cell population positive for amarker refers to a percentage of cells that exhibit the marker, whichmay be at least 50% of the cells, 55% of the cells, 60% of the cells,65% of the cells, 70% of the cells, 75% of the cells, 80% of the cells,85% of the cells, 90% of the cell, 95% of the cells, or 100% of thecells, or any percentage between 50% and 100% when compared to areference T cell population.

Immunomagnetic selection methods may also be used to purify T cellsubpopulations using commercially available clinical grade antibody beadconjugates using a CliniMACS device (see, e.g., Terakura et al., 2012,Blood 119:72-82; Wang et al., 2012, J. Immunother. 35:689-701). Forexample, to isolate human CD8+ T_(CM) cells, CD4+, CD14+, and CD45RA+cells are removed from peripheral blood mononuclear cells by depletionwith antibody conjugated paramagnetic beads, and then the CD62L+fraction from the remaining cells is positively selected with ananti-CD62L labeled bead to enrich for the CD45RO+, CD62L+, CD8+ T_(CM)subpopulation. The enriched CD8+ T_(CM) subpopulation can be activatedwith anti-CD3/CD28 beads or with antigen, modified with tumor-specificCAR using retroviral or lentiviral vectors, and expanded for use incellular immunotherapy (see, e.g., Terakura et al., supra; Wang et al.,supra).

Alternatively, T cell subsets may be selected using low-affinity Fabfragments fused to Strep-tag II. A Fab monomers do not have sufficientbinding affinity for stable binding to a target antigen on the cellsurface. However, when multimerized on a StrepTactin bead, thesereagents stably bind a target cell and enable selection based on cellsurface marker specificity. A Fab multimer binding can be rapidlyreversed by the addition of excess D-biotin, which has a higher affinityfor StrepTactin and disrupts the binding between a Strep-tag on aFab-fragment and a Strep-Tactin “backbone.” Fab monomers cannot maintainstable binding a the cell. This “Fab-Streptamers” technology allows forserial positive enrichment of T cells based on multiple cell surfacemarkers and can be used to select any desired T cell subset (see, e.g.,Stemberger et al., PluS One 7:e35793, 2012).

Bulk CD8+ T cells can be obtained by using standard methods. In someembodiments, bulk CD8+ T cells are further sorted into naïve, centralmemory, and effector T cells by identifying certain cell surface markersthat are associated with each of those types of CD8+ T cells. In certainembodiments, memory T cells are present in both CD62L+ and CD62L−subsets of CD8+ peripheral blood lymphocytes. For example, PBMCs can besorted into CD62L−CD8+ and CD62L+CD8+ fractions after staining withanti-CD8 and anti-CD62L antibodies. In some embodiments, expression ofphenotypic markers of CD8+ central memory T cells include CD45RO, CD62L,CCR7, CD28, CD3, or CD127 or are negative for granzyme B. In someembodiments, central memory T cells are CD45RO+, CD62L+, CD8+ T cells.In some embodiments, CD8+ effector T cells are negative for or havereduced expression of CD62L, CCR7, CD28, or CD127, or are positive foror have increased expression of granzyme B or perforin, as compared toCD8+ central memory T cells. In some embodiments, naïve CD8+ T cells arecharacterized by the expression of phenotypic markers of naïve T cellsincluding CD62L, CCR7, CD28, CD3, CD127, or CD45RA.

Bulk CD4+ lymphocytes can be obtained by standard methods. In someembodiments, bulk CD4+ T cells are further sorted into naïve, centralmemory, and effector cells by identifying cell populations that havecertain cell surface markers. In some embodiments, naïve CD4+ Tlymphocytes are CD45RO−, CD45RA+, CD62L+, CD4+ T cell. In someembodiments, central memory CD4+ cells are CD62L positive and CD45ROpositive. In some embodiments, effector CD4+ cells are CD62L or CD45ROnegative or have reduced expression of CD62L or CD45RO as compared tocentral memory CD4+ cells.

Populations of CD4+ and CD8+ having TCRs that are antigen specific canbe obtained by stimulating naïve or antigen-specific T lymphocytes withantigen. For example, T cell clones having antigen-specific TCRs can begenerated against, for example, Cytomegalovirus antigens by isolating Tcells from infected subjects and stimulating the cells in vitro with thesame antigen. Naïve T cells may also be used by exposing them to peptideantigens presented in the context of an antigen presenting cell or apeptide-MHC complex. Any number of antigens from tumor cells, cancercells, or pathogenic agents may be utilized. Examples of such antigensinclude HIV antigens, Hepatitis C Virus (HCV) antigens, Hepatitis BVirus (HBV) antigens, Cytomegalovirus (CMV) antigens, EBV antigens,parasitic antigens, and tumor antigens, such as orphan tyrosine kinasereceptor ROR1, EGFR, EGFRvIII, GD2, GD3, HPV E6, HPV E7, Her2, L1-CAM,Lewis A, Lewis Y, MUC1, MUC16, PSMA, CD19, CD20, CD22, CD56, CD23, CD24,CD37, CD30, CD33, CD38, CD56, CD123, CA125, c-MET, FcRH5, WT1, folatereceptor α, VEGF-α, VEGFR1, VEGFR2, IL-13Rα2, IL-11Rα, MAGE-A1, PSA,ephrin A2, ephrin B2, NKG2D ligands, NY-ESO-1, TAG-72, mesothelin, CEA,or the like. Such T cells having antigen-specific TCRs may be furthermodified to contain a fusion protein as described herein, wherein thefusion protein is specific for the same antigen, specific for adifferent epitope on the same antigen, or specific for a differentantigen. In any of these embodiments, the CD4+ T cells and the CD8+ Tcells will contain different CARs, and in particular the intracellularsignaling components of the CARs will be distinct.

Methods of preparing and modifying T cells to express fusion proteins ofthis disclosure, confirming fusion protein modified T cell activity,expanding fusion protein modified T cell populations are known in theart and are described, for example, in Hollyman et al., 2009, J.Immunother. 32:169-180; PCT Publication No. WO 2012/079000; U.S. Pat.No. 8,802,374; Brentjens et al., Blood 118:4817-4828; 2011; U.S. PatentPublication No. US 2014/0271635.

Uses

The present disclosure provides methods of treating a disease,condition, or disorder in a subject comprising: administering any of thefusion proteins described herein to the subject. In embodiments, methodsof the present disclosure include methods of reducing the number ofB-cells or treating a disease or disorder associated with aberrantB-cell activity in a subject. Another embodiment provides a method oftreating a disease, condition, or disorder a subject comprisinganalyzing a biological sample of the subject for the presence of anantigen associated with the disease, condition, or disorder andadministering a fusion protein described herein, wherein the fusionprotein specifically binds to the antigen. In some embodiments, theantigen associated with the disease, condition, or disorder is a tumorassociated antigen.

Diseases, conditions, or disorders that may be treated with compositionsand methods as described in the present disclosure include cancer andimmune diseases (e.g., autoimmune). For example, in certain embodiments,a CD20-expressing cell comprises B-cells. In further embodiments, thedisease or disorder associated with CD20 expression is in B-cells oraberrant B cell activity, such as B-cell-related cancers. Adoptiveimmune and gene therapy are promising treatments for various types ofcancer (Morgan et al., Science 314:126, 2006; Schmitt et al., Hum. GeneTher. 20:1240, 2009; June, J. Clin. Invest. 117:1466, 2007).

A wide variety of cancers, including solid tumors and leukemias areamenable to the compositions and methods disclosed herein. Exemplarytypes of cancer that may be treated include adenocarcinoma of thebreast, prostate, and colon; all forms of bronchogenic carcinoma of thelung; myeloid leukemia; melanoma; hepatoma; neuroblastoma; papilloma;apudoma; choristoma; branchioma; malignant carcinoid syndrome; carcinoidheart disease; and carcinoma (e.g., Walker, basal cell, basosquamous,Brown-Pearce, ductal, Ehrlich tumor, Krebs 2, Merkel cell, mucinous,non-small cell lung, oat cell, papillary, scirrhous, bronchiolar,bronchogenic, squamous cell, and transitional cell). Additional types ofcancers that may be treated include histiocytic disorders; malignanthistiocytosis; leukemia; Hodgkin's disease; immunoproliferative small;non-Hodgkin's lymphoma; plasmacytoma; reticuloendotheliosis; melanoma;chondroblastoma; chondroma; chondrosarcoma; fibroma; fibrosarcoma; giantcell tumors; histiocytoma; lipoma; liposarcoma; mesothelioma; myxoma;myxosarcoma; osteoma; osteosarcoma; chordoma; craniopharyngioma;dysgerminoma; hamartoma; mesenchymoma; mesonephroma; myosarcoma;ameloblastoma; cementoma; odontoma; teratoma; thymoma; trophoblastictumor. Further, the following types of cancers are also contemplated asamenable to treatment: adenoma; cholangioma; cholesteatoma; cyclindroma;cystadenocarcinoma; cystadenoma; granulosa cell tumor; gynandroblastoma;hepatoma; hidradenoma; islet cell tumor; Leydig cell tumor; papilloma;sertoli cell tumor; theca cell tumor; leimyoma; leiomyosarcoma;myoblastoma; myomma; myosarcoma; rhabdomyoma; rhabdomyosarcoma;ependymoma; ganglioneuroma; glioma; medulloblastoma; meningioma;neurilemmoma; neuroblastoma; neuroepithelioma; neurofibroma; neuroma;paraganglioma; paraganglioma nonchromaffin. The types of cancers thatmay be treated also include angiokeratoma; angiolymphoid hyperplasiawith eosinophilia; angioma sclerosing; angiomatosis; glomangioma;hemangioendothelioma; hemangioma; hemangiopericytoma; hemangiosarcoma;lymphangioma; lymphangiomyoma; lymphangiosarcoma; pinealoma;carcinosarcoma; chondrosarcoma; cystosarcoma phyllodes; fibrosarcoma;hemangiosarcoma; leiomyosarcoma; leukosarcoma; liposarcoma;lymphangiosarcoma; myosarcoma; myxosarcoma; ovarian carcinoma;rhabdomyosarcoma; sarcoma; neoplasms; nerofibromatosis; and cervicaldysplasia.

Exemplifying the variety of hyperproliferative disorders amenable to thecompositions and methods disclosed herein described herein are disordersor diseases associated with CD20 expression, such as aberrant B-cellactivity, including B-cell cancers, such as B-cell lymphomas (such asvarious forms of Hodgkin's disease, non-Hodgkins lymphoma (NHL) orcentral nervous system lymphomas), leukemias (such as acutelymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), hairycell leukemia, B cell blast transformation of chronic myeloid leukemia)and myelomas (such as multiple myeloma). Additional B cell cancersinclude small lymphocytic lymphoma (SLL), Waldenström'smacroglobulinemia, CD37+ dendritic cell lymphoma, B-cell prolymphocyticleukemia, lymphoplasmacytic lymphoma, splenic marginal zone lymphoma,plasma cell myeloma, solitary plasmacytoma of bone, extraosseousplasmacytoma, extra-nodal marginal zone B-cell lymphoma ofmucosa-associated (MALT) lymphoid tissue, nodal marginal zone B-celllymphoma, follicular lymphoma, mantle cell lymphoma, diffuse largeB-cell lymphoma, mediastinal (thymic) large B-cell lymphoma, precursorB-lymphoblastic lymphoma, immunoblastic large cell lymphoma,intravascular large B-cell lymphoma, primary effusion lymphoma,Burkitt's lymphoma/leukemia, B-cell proliferations of uncertainmalignant potential, lymphomatoid granulomatosis, and post-transplantlymphoproliferative disorder. In certain embodiments, the compositionsand methods of this disclosure can be used treat non-B-cell disorders ordiseases associated with CD20 expression, including multiple myeloma,melanoma, multiple myeloma of stem cells and melanoma of stem cells.

Inflammatory and autoimmune diseases amenable to the compositions andmethods disclosed herein include arthritis, rheumatoid arthritis,juvenile rheumatoid arthritis, osteoarthritis, polychondritis, psoriaticarthritis, psoriasis, dermatitis, idiopathic inflammatory myopathy,polymyositis/dermatomyositis, inclusion body myositis, inflammatorymyositis, toxic epidermal necrolysis, systemic scleroderma andsclerosis, CREST syndrome, inflammatory bowel disease, Crohn's disease,Grave's disease, ulcerative colitis, respiratory distress syndrome,adult respiratory distress syndrome (ARDS), meningitis, encephalitis,uveitis, colitis, glomerulonephritis, allergic conditions, eczema,asthma, conditions involving infiltration of T cells and chronicinflammatory responses, atherosclerosis, autoimmune myocarditis,leukocyte adhesion deficiency, systemic lupus erythematosus (SLE),subacute cutaneous lupus erythematosus, discoid lupus, lupus myelitis,lupus cerebritis, juvenile onset diabetes, multiple sclerosis, allergicencephalomyelitis, neuromyelitis optica, rheumatic fever, Sydenham'schorea, immune responses associated with acute and delayedhypersensitivity mediated by cytokines and T-lymphocytes, tuberculosis,sarcoidosis, granulomatosis including Wegener's granulomatosis andChurg-Strauss disease, agranulocytosis, vasculitis (includinghypersensitivity vasculitis/angiitis, ANCA and rheumatoid vasculitis),aplastic anemia, Diamond Blackfan anemia, immune hemolytic anemiaincluding autoimmune hemolytic anemia (AIHA), pernicious anemia, purered cell aplasia (PRCA), Factor VIII deficiency, hemophilia A,autoimmune neutropenia, pancytopenia, leukopenia, diseases involvingleukocyte diapedesis, central nervous system (CNS) inflammatorydisorders, multiple organ injury syndrome, myasthenia gravis,antigen-antibody complex mediated diseases, anti-glomerular basementmembrane disease, anti-phospholipid antibody syndrome, allergicneuritis, Behcet disease, Castleman's syndrome, Goodpasture's syndrome,Lambert-Eaton Myasthenic Syndrome, Reynaud's syndrome, Sjorgen'ssyndrome, Stevens-Johnson syndrome, solid organ transplant rejection,graft versus host disease (GVHD), bullous pemphigoid, pemphigus,autoimmune polyendocrinopathies, seronegative spondyloarthropathies,Reiter's disease, stiff-man syndrome, giant cell arteritis, immunecomplex nephritis, IgA nephropathy, IgM polyneuropathies or IgM mediatedneuropathy, idiopathic thrombocytopenic purpura (ITP), thromboticthrobocytopenic purpura (TTP), Henoch-Schonlein purpura, autoimmunethrombocytopenia, autoimmune disease of the testis and ovary includingautoimmune orchitis and oophoritis, primary hypothyroidism; autoimmuneendocrine diseases including autoimmune thyroiditis, chronic thyroiditis(Hashimoto's Thyroiditis), subacute thyroiditis, idiopathichypothyroidism, Addison's disease, Grave's disease, autoimmunepolyglandular syndromes (or polyglandular endocrinopathy syndromes),Type I diabetes mellitus, also referred to as insulin-dependent diabetesmellitus (IDDM), and Sheehan's syndrome; autoimmune hepatitis, lymphoidinterstitial pneumonitis (HIV), bronchiolitis obliterans(non-transplant), non-specific interstitial pneumonia (NSIP),Guillain-Barré Syndrome, large vessel vasculitis (including polymyalgiarheumatica and giant cell (Takayasu's) arteritis), medium vesselvasculitis (including Kawasaki's disease and polyarteritis nodosa),polyarteritis nodosa (PAN) ankylosing spondylitis, Berger's disease (IgAnephropathy), rapidly progressive glomerulonephritis, primary biliarycirrhosis, Celiac sprue (gluten enteropathy), cryoglobulinemia,cryoglobulinemia associated with hepatitis, amyotrophic lateralsclerosis (ALS), coronary artery disease, familial Mediterranean fever,microscopic polyangiitis, Cogan's syndrome, Whiskott-Aldrich syndromeand thromboangiitis obliterans.

Subjects that can be treated by the present invention are, in general,human and other primate subjects, such as monkeys and apes forveterinary medicine purposes. The subjects can be male or female and canbe any suitable age, including infant, juvenile, adolescent, adult, andgeriatric subjects.

Fusion proteins of the present disclosure may be formulated foradministration in any suitable manner, as understood by persons skilledin the art. A CD20-specific fusion protein (e.g., a CAR) of thisdisclosure (or fusion protein specific for a different target) may beadministered to a subject in cell-bound form (e.g., ex vivo modificationof a target cell population (mature T cells (e.g., CD8⁺ or CD4⁺ T cells)or other cells of T cell lineage)). In a particular embodiment, cells ofT cell lineage expressing CD20-specific fusion proteins of thisdisclosure (or fusion protein specific for a different target)administered to a subject are syngeneic, allogeneic, or autologous cellsto the subject. In some embodiments, cells comprising fusion proteins ofthis disclosure are prepared by harvesting cells (from a biologicalsample, tissue, or culture medium), washing, concentrating, andformulating in a medium and container system suitable foradministration.

The present disclosure provides compositions comprising cells expressingfusion proteins as disclosed herein and a pharmaceutically acceptablecarrier, diluents, or excipient. Suitable excipients include water,saline, dextrose, glycerol, or the like and combinations thereof. Inembodiments, compositions comprising cells expressing fusion proteins asdisclosed herein further comprise a suitable infusion media. Suitableinfusion media can be any isotonic medium formulation, typically normalsaline, Normosol R (Abbott) or Plasma-Lyte A (Baxter), 5% dextrose inwater, Ringer's lactate can be utilized. An infusion medium can besupplemented with human serum albumin or other human serum components.

In other embodiments, CD20-specific fusion proteins of this disclosure(or fusion protein specific for a different target) may be administeredto a subject in soluble form. For example, soluble TCRs are known in theart (see, e.g., Molloy et al., Curr. Opin. Pharmacol. 5:438, 2005; U.S.Pat. No. 6,759,243).

Fusion proteins of this disclosure, or cells including the same, may beadministered in a manner appropriate to the disease, condition, ordisorder to be treated as determined by persons skilled in the medicalart. In any of the above embodiments, a cell comprising a fusion proteinas described herein is administered intravenously, intraperitoneally,intratumorly, into the bone marrow, into a lymph node, or intocerebrospinal fluid. In some embodiments, cells comprising a fusionprotein of the present disclosure are delivered to the site of a tumor.

An appropriate dose, suitable duration, and frequency of administrationof the compositions will be determined by such factors as a condition ofthe patient; size, type, and severity of the disease, condition, ordisorder; particular form of the active ingredient; and the method ofadministration.

In any of the above embodiments, methods of the present disclosurecomprise administering a therapeutically effective amount of a host cellexpressing a fusion protein of the present disclosure or a host cellexpressing a fusion of this disclosure. A therapeutically effectiveamount of cells in a composition is at least one cell (for example, onefusion protein modified CD8+ T cell subpopulation; one fusion proteinmodified CD4+ T cell subpopulation) or is more typically greater than10² cells, for example, up to 10⁶, up to 10⁷, up to 10⁸ cells, up to 10⁹cells, or more than 10¹⁰ cells. In certain embodiments, the cells areadministered in a range from about 10⁶ to about 10¹⁰ cells/m²,preferably in a range of about 10⁵ to about 10⁹ cells/m². The number ofcells will depend upon the ultimate use for which the composition isintended as well the type of cells included therein. For example, cellsmodified to contain a fusion protein specific for a particular antigenwill comprise a cell population containing at least 30%, 35%, 40%, 45%,50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more of such cells.For uses provided herein, cells are generally in a volume of a liter orless, 500 mls or less, 250 mls or less, or 100 mls or less. Inembodiments, the density of the desired cells is typically greater than10⁴ cells/ml and generally is greater than 10⁷ cells/ml, generally 10⁸cells/ml or greater. The cells may be administered as a single infusionor in multiple infusions over a range of time. A clinically relevantnumber of immune cells can be apportioned into multiple infusions thatcumulatively equal or exceed 10⁶, 10⁷, 10⁸, 10⁹, 10¹⁰, or 10¹¹ cells.

In some embodiments, methods of the present disclosure compriseadministering a host cell expressing a CAR of this disclosure that isfully human or humanized. In any of the embodiments described herein,methods of the present disclosure comprise administering a host cellexpressing a CAR that has a scFv from an anti-CD20 antibody or a scTCRfrom a TCR specific for a CD20 antigen. In any of the embodimentsdescribed herein, methods of the present disclosure compriseadministering a host cell expressing a CAR that comprises a scFv from1.5.3, 1F5, Leu16, rituximab, ofatumumab, veltuzumab, ocrelizumab,ublituximab, or any combination thereof. In any of the aboveembodiments, methods of the present disclosure comprise administering ahost cell expressing a CAR that comprises a linker module comprising anIgG1 hinge, an IgG4 hinge, or any combination thereof. In any of theembodiments described herein, methods of the present disclosure compriseadministering a host cell expressing a CAR that comprises a linkermodule comprising an IgG1 CH2 region with a N297Q mutation, an IgG4 CH2region, an IgG1 CH3 region, an IgG4 CH3 region, or any combinationthereof. In any of the embodiments of this disclosure, methods of thepresent disclosure comprise administering a host cell expressing a CARthat comprises a glycine-serine linker module or glycine-serine variableregion linker. In any of the embodiments described herein, methods ofthe present disclosure comprise administering a host cell expressing aCAR that comprises a hydrophobic portion comprised of a CD28transmembrane domain. In any of the embodiments described herein,methods of the present disclosure comprise administering a host cellexpressing a CAR that comprises an intracellular domain comprising adomain from CD3ζ, 4-1BB, CD28, or any combination thereof. In any of theabove embodiments, methods of the present disclosure compriseadministering a CAR that comprises junction amino acids between adjacentdomains, motifs, regions, modules, or fragments.

In any of the embodiments described herein, methods of this disclosurecomprise administering to a subject a host cell comprising aheterologous nucleic acid molecule encoding a fusion protein, the fusionprotein comprising an extracellular component and an intracellularcomponent connected by a hydrophobic portion, wherein the extracellularcomponent comprises a binding domain that specifically binds CD20 andthe intracellular component comprises an effector domain, wherein theencoded fusion protein (e.g., CAR) is at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, at least 99.5%, or 100% identicalto 1.5.3-NQ-28-BB-z (SEQ ID NO.:26); 1.5.3-NQ-28-z (SEQ ID NO.:27);1.5.3-NQ-BB-z (SEQ ID NO.:28); 1.5.3-NQ-z (SEQ ID NO.:29); Leu16-28-BB-z(SEQ ID NO.:30); Leu16-28-z (SEQ ID NO.:31); 1F5-NQ-28-BB-z (SEQ IDNO.:32); 1F5-NQ-28-z (SEQ ID NO.:33); or 1F5-NQ-BB-z (SEQ ID NO.:34). Infurther embodiments, methods of the present disclosure compriseadministering to a subject a host cell comprising a heterologous nucleicacid molecule encoding a fusion protein, the fusion protein comprisingan extracellular component and an intracellular component connected by ahydrophobic portion, wherein the extracellular component comprises abinding domain that specifically binds CD20 and the intracellularcomponent comprises an effector domain, wherein the encoded fusionprotein (e.g., CAR) comprises or consists of an amino acid sequence ofany one of SEQ ID NOS.:26-34. In any of these embodiments, the host cellis a T cell, wherein the T cells bulk CD4+ T cells, bulk CD8+ T cells,CD4+ central memory T cells, CD8+ central memory T cells or acombination of CD4+ central memory (T_(CM)) and CD4+ naïve (T_(N)) Tcells. The CAR-modified CD4+ T cells and CAR-modified CD8+ T cells canbe mixed in a ratio of 3:1 to 1:1 to 1:3 before administration to asubject, or can be administered to a subject separately at the same orsimilar ratios.

In still further embodiments, methods of this disclosure compriseadministering to a subject a host cell comprising a heterologous nucleicacid molecule encoding a fusion protein, the fusion protein comprisingan extracellular component and an intracellular component connected by ahydrophobic portion, wherein the extracellular component comprises abinding domain that specifically binds CD20 and the intracellularcomponent comprises an effector domain, wherein the fusion protein(e.g., CAR) is encoded by a polynucleotide having at least 60%, at least70%, at least 75%, at least 80%, at least 85%, at least 90%, at least95%, or 100% identity to a nucleic acid molecule sequence of any one ofSEQ ID NOS.:44-52. In related embodiments, methods compriseadministering to a subject a host cell comprising a heterologous nucleicacid molecule encoding a fusion protein, the fusion protein comprisingan extracellular component and an intracellular component connected by ahydrophobic portion, wherein the extracellular component comprises abinding domain that specifically binds CD20 and the intracellularcomponent comprises an effector domain, wherein the fusion protein(e.g., CAR) is encoded by a polynucleotide comprising or consisting of asequence of any one of SEQ ID NOS.:44-52. In any of these embodiments,the host cell is a T cell, wherein the T cells bulk CD4+ T cells, bulkCD8+ T cells, CD4+ central memory T cells, CD8+ central memory T cellsor a combination of CD4+ central memory (T_(CM)) and CD4+ naïve (T_(N))T cells. The CAR-modified CD4+ T cells and CAR-modified CD8+ T cells canbe mixed in a ratio of 3:1 to 1:1 to 1:3 before administration to asubject, or can be administered to a subject separately at the same orsimilar ratios.

Optionally, a host cell comprising any vector of this disclosure thatcontains a polynucleotide that encodes a fusion protein of thisdisclosure, for use in the methods described herein, can also encode atransduction marker (e.g., tCD19), which may also include aself-cleaving peptide so that the transduction marker and CAR areseparated into separate molecules—a CAR and a transduction marker. Incertain embodiments, a host cell (e.g., T cell) comprises a vector thatcontains a polynucleotide encoding a self-cleaving peptide disposedbetween a CD20-specific CAR and a tCD19 transduction marker, whichpolynucleotide is at least 60%, at least 70%, at least 75%, at least80%, at least 85%, at least 90%, at least 95%, or 100% identical to anucleic acid molecule sequence of any one of SEQ ID NOS.:53-61. Infurther embodiments, a host cell (e.g., T cell) comprises a vector thatcontains a polynucleotide encoding a self-cleaving peptide disposedbetween a CD20-specific CAR and a tCD19 transduction marker andcomprises or consists of a nucleic acid molecule sequence of any one ofSEQ ID NOS.:53-61.

Accordingly, in any of the methods disclosed herein a host cellcomprises an isolated polynucleotide encoding a fusion protein capableof specifically binding CD20, wherein the polynucleotide: (a) is atleast 80% identical to a polynucleotide sequence of any one of SEQ IDNOS.:53-56; (b) is at least 80% identical to a polynucleotide sequenceof any one of SEQ ID NOS.:44-47; (c) comprises a polynucleotide sequenceof any one of SEQ ID NOS.:53-56; (d) comprises a polynucleotide sequenceof any one of SEQ ID NOS.:44-47; (e) consists of a polynucleotidesequence of any one of SEQ ID NOS.:53-56; or (f) consists of apolynucleotide sequence of any one of SEQ ID NOS.:44-47.

In certain embodiments, the host cell used in the methods comprises afusion protein that consists of or comprises an amino acid sequencewherein the fusion protein: (a) is at least 90% identical to a maturefusion protein, wherein the mature fusion protein comprises an aminoacid sequence of any one of SEQ ID NOS.:26-29 and 35-38 and 32-34 withthe tCD19 transduction marker removed; (b) is comprised of a maturefusion protein, wherein the mature fusion protein comprises an aminoacid sequence of any one of SEQ ID NOS.: 35-38 with the tCD19transduction marker removed; (c) consists of a mature fusion protein,wherein the mature fusion protein comprises an amino acid sequence ofany one of SEQ ID NOS.: 35-38 with the tCD19 transduction markerremoved; (d) is at least 90% identical to an amino acid sequence of anyone of SEQ ID NOS.:26 29; (e) is comprised of an amino acid sequence ofany one of SEQ ID NOS.:26 29; (f) consists of an amino acid sequence ofany one of SEQ ID NOS.:26 29.

In certain embodiments, a host cell used in the methods comprises aheterologous polynucleotide as disclosed herein and is capable ofexpressing the fusion protein.

In certain embodiments, a host cell used in the methods comprises aheterologous polynucleotide encoding a fusion protein comprising abinding domain, wherein the binding domain is: (a) a 1.5.3 scFvcomprising an amino acid sequence that is at least 90% identical to anamino acid sequence of SEQ ID NO.:64, wherein each CDR of the scFvcomprises zero changes or at most one, two, three, four, five or sixchanges as compared to the corresponding CDR of a parent monoclonalantibody or fragment or derivative thereof that specifically binds toCD20, provided that scFv containing one or more modified CDRsspecifically binds CD20 with an affinity similar to the wild type scFvor corresponding antibody; (b) a 1.5.3 scFv comprising or consisting ofan amino acid sequence of SEQ ID NO.:64; (c) a 1F5 scFv comprising anamino acid sequence that is at least 90% identical to an amino acidsequence of SEQ ID NO.:66, wherein each CDR of the scFv comprises zerochanges or at most one, two, three, four, five or six changes ascompared to the corresponding CDR of a parent monoclonal antibody orfragment or derivative thereof that specifically binds to CD20, providedthat scFv containing one or more modified CDRs specifically binds CD20with an affinity similar to the wild type scFv or correspondingantibody; (d) a 1F5 scFv comprising or consisting of an amino acidsequence of SEQ ID NO.:66; (e) a Leu16 scFv comprising an amino acidsequence that is at least 90% identical to an amino acid sequence of SEQID NO.:65, wherein each CDR of the scFv comprises zero changes or atmost one, two, three, four, five or six changes as compared to thecorresponding CDR of a parent monoclonal antibody or fragment orderivative thereof that specifically binds to CD20, provided that scFvcontaining one or more modified CDRs specifically binds CD20 with anaffinity similar to the wild type scFv or corresponding antibody; or (f)a Leu16 scFv comprising or consisting of an amino acid sequence of SEQID NO.:65.

In certain embodiments, a host cell used in the methods comprises aheterologous polynucleotide encoding a fusion protein comprising anscFv, wherein the scFv is encoded by: (a) a polynucleotide having atleast 80% identity to a nucleic acid molecule sequence of SEQ ID NO.:67,wherein polynucleotide sequences encoding each CDR of a scFv compriseszero changes or at most one to six nucleotide changes, as compared to apolynucleotide encoding a parent scFv from a monoclonal antibody thatspecifically binds to CD20, provided that scFv containing one or moremodified CDRs specifically binds CD20 with an affinity similar to thewild type scFv or corresponding antibody; (b) a polynucleotidecomprising or consisting of a nucleic acid molecule sequence of SEQ IDNO.:67; (c) a polynucleotide having at least 80% identity to a nucleicacid molecule sequence of SEQ ID NO.:69, wherein polynucleotidesequences encoding each CDR of a scFv comprises zero changes or at mostone to six nucleotide changes, as compared to a polynucleotide encodinga parent scFv from a monoclonal antibody that specifically binds toCD20, provided that scFv containing one or more modified CDRsspecifically binds CD20 with an affinity similar to the wild type scFvor corresponding antibody; (d) a polynucleotide comprising or consistingof a nucleic acid molecule sequence of SEQ ID NO.:69; (e) apolynucleotide having at least 80% identity to a nucleic acid moleculesequence of SEQ ID NO.:68, wherein polynucleotide sequences encodingeach CDR of a scFv comprises zero changes or at most one to sixnucleotide changes, as compared to a polynucleotide encoding a parentscFv from a monoclonal antibody that specifically binds to CD20,provided that scFv containing one or more modified CDRs specificallybinds CD20 with an affinity similar to the wild type scFv orcorresponding antibody; or (f) a polynucleotide comprising or consistingof a nucleic acid molecule sequence of SEQ ID NO.:68.

In certain embodiments, a host cell used in the methods comprises aheterologous polynucleotide encoding a fusion protein, wherein thefusion protein is a chimeric antigen receptor and comprises or consistsof an amino acid sequence that is at least 90% identical to an aminoacid sequence of any one of SEQ ID NOS.:26-43.

In certain embodiments, a host cell used in the methods comprises aheterologous polynucleotide encoding a fusion protein comprising ahydrophobic portion, wherein the hydrophobic portion is a transmembranedomain. In certain embodiments, the hydrophobic portion is a CD4, CD8,CD28 or CD27 transmembrane domain.

In certain embodiments, a host cell used in the methods comprises aheterologous polynucleotide encoding a fusion protein comprising aneffector domain or functional portion thereof, wherein the effectordomain or functional portion thereof is a 4-1BB (CD137), CD3ε, CD3δ,CD3ζ, CD25, CD27, CD28, CD79A, CD79B, CARD11, DAP10, FcRα, FcRβ, FcRγ,Fyn, HVEM, ICOS, Lck, LAG3, LAT, LRP, NKG2D, NOTCH1, NOTCH2, NOTCH3,NOTCH4, OX40 (CD134), ROR2, Ryk, SLAMF1, Slp76, pTα, TCRα, TCRβ, TRIM,Zap70, PTCH2, or any combination thereof.

In certain embodiments, a host cell used in the methods comprises aheterologous polynucleotide encoding a fusion protein comprising anintracellular component, wherein the intracellular component comprises:(a) a CD3ζ effector domain or functional portion thereof, a CD28costimulatory domain or functional portion thereof and a 4-1BB (CD137)costimulatory domain or portion thereof; (b) a CD3ζ effector domain orfunctional portion thereof, a CD28 costimulatory domain or functionalportion thereof and a OX40 (CD134) costimulatory domain or portionthereof; (c) a CD3ζ effector domain or functional portion thereof, aCD27 costimulatory domain or functional portion thereof and a 4-1BB(CD137) costimulatory domain or portion thereof; (d) a CD3ζ effectordomain or functional portion thereof, a CD27 costimulatory domain orfunctional portion thereof and a OX40 (CD134) costimulatory domain orportion thereof; (e) a CD3ζ effector domain or functional portionthereof, a CD27 costimulatory domain or functional portion thereof and aCD28 costimulatory domain or portion thereof; or (f) a CD3ζ effectordomain or functional portion thereof, a 4-1BB (CD137) costimulatorydomain or functional portion thereof and a OX40 (CD134) costimulatorydomain or portion thereof.

Compositions of this disclosure may also be administered simultaneouslywith, prior to, or after administration of one or more other therapeuticagents. Such combination therapy includes administration of a singledosage formulation which contains a fusion protein of this disclosureand one or more additional active agents, as well as administration of afusion protein of this disclosure and each active agent in its ownseparate dosage formulation. For example, a fusion protein of thisdisclosure and another active agent can be administered to a subjecttogether in a single infusion dosage composition, or each agent can beadministered in separate infusion dosage formulations. Where separatedosage formulations are used, a fusion protein of this disclosure andone or more additional active agents can be administered at the sametime, i.e., simultaneously, at essentially the same time, i.e.,concurrently, or at separately staggered times, i.e., sequentially;combination therapy is understood to include all these regimens.

The present disclosure provides pharmaceutical compositions comprisingCD20-specific binding molecules, cells expressing fusion proteins asdisclosed herein or both, and a pharmaceutically acceptable carrier,diluents, or excipient. In certain embodiments, the CD20-specificbinding molecule is an antibody. In such embodiments, a CD20-specificantibody can be rituximab, ofatumumab, ocrelizumab, obinutuzumab,ublituximab, veltuzumab, ibritumomab tiuxetan, tositumomab, or anycombination thereof.

In certain embodiments, a method of treating a disease or disorderassociated with CD20 expression comprises administering to a subjecthaving or suspected of having a disease or disorder associated with CD20expression a therapeutically effective amount of a host cell comprisinga heterologous nucleic acid molecule encoding a fusion protein, thefusion protein comprising an extracellular component and anintracellular component connected by a hydrophobic portion, wherein theextracellular component comprises a binding domain that specificallybinds CD20 and the intracellular component comprises an effector domain,and optionally administering a therapeutically effective amount of aCD20-specific binding molecule, chemotherapeutic or inhibitor of animmunosuppression component. In further embodiments, the method reducesthe number of B-cells or treats a disease or disorder associated withaberrant B-cell activity.

Thus, in certain embodiments, provided are methods of treating a diseaseor disorder associated with CD20 expression, comprising administering toa subject having or suspected of having a disease or disorder associatedwith CD20 expression a therapeutically effective amount of a host cellcomprising a heterologous nucleic acid molecule encoding a fusionprotein comprised of an amino acid sequence that is at least 90%identical to an amino acid sequence of any one of SEQ ID NOS.: 26-29 and32-38, and 41-43, and optionally administering a CD20-specific bindingmolecule, a chemotherapeutic, an inhibitor of an immunosuppressioncomponent, or combinations thereof. In further embodiments, the methodreduces the number of B-cells or treats a disease or disorder associatedwith aberrant B-cell activity.

In some embodiments, compositions as described herein are administeredwith chemotherapeutic agents or immune modulators (e.g.,immunosuppressants, or inhibitors of immunosuppression components, suchas immune checkpoint inhibitors). Immune checkpoint inhibitors includeinhibitors of CTLA-4, A2AR, B7-H3, B7-H4, BTLA, HVEM, GALS, IDO, KIR,LAG-3, PD-1, PD-L1, PD-L2, Tim-3, VISTA, TIGIT, LAIR1, CD160, 2B4, TGFRbeta, CEACAM-1, CEACAM-3, CEACAM-5, CD244, or any combination thereof.An inhibitor of an immune checkpoint molecule can be an antibody orantigen binding fragment thereof, a fusion protein, a small molecule, anRNAi molecule, (e.g., siRNA, shRNA, or miRNA), a ribozyme, an aptamer,or an antisense oligonucleotide. A chemotherapeutic can be a B-Rafinhibitor, a MEK inhibitor, a VEGF inhibitor, a VEGFR inhibitor, atyrosine kinase inhibitor, an anti-mitotic agent, or any combinationthereof.

In any of the embodiments herein, a method of treating a disease ordisorder associated with CD20 expression comprises administering to asubject having or suspected of having a disease or disorder associatedwith CD20 expression a therapeutically effective amount of a host cellcomprising a heterologous nucleic acid molecule encoding a fusionprotein as disclosed herein, and a therapeutically effective amount ofan inhibitor of an immunosuppression component, such as an immunecheckpoint inhibitor. In some embodiments, an immune checkpointinhibitor is an inhibitor of CTLA-4, A2AR, B7-H3, B7-H4, BTLA, HVEM,GAL9, IDO, KIR, LAG-3, PD-1, PD-L1, PD-L2, Tim-3, VISTA, TIGIT, LAIR1,CD160, 2B4, TGFR beta, CEACAM-1, CEACAM-3, CEACAM-5, CD244, or anycombination thereof.

Accordingly, in certain embodiments, this disclosure provides methods oftreating a disease or disorder associated with CD20 expression,comprising administering to a subject having or suspected of having adisease or disorder associated with CD20 expression a therapeuticallyeffective amount of a host cell comprising a heterologous nucleic acidmolecule encoding a fusion protein having an amino acid sequence that isat least 90% identical to an amino acid sequence of any one of SEQ IDNOS.: 26-29 and 32-38, and 41-43, and a therapeutically effective amountof an inhibitor of an immunosuppression component, such as an immunecheckpoint inhibitor. In some embodiments, an immune checkpointinhibitor is an inhibitor of CTLA-4, A2AR, B7-H3, B7-H4, BTLA, HVEM,GAL9, IDO, KIR, LAG-3, PD-1, PD-L1, PD-L2, Tim-3, VISTA, TIGIT, LAIR1,CD160, 2B4, TGFR beta, CEACAM-1, CEACAM-3, CEACAM-5, CD244, or anycombination thereof. In some embodiments, an immune checkpoint inhibitoris selected from (a) an antibody specific for PD-1, such as pidilizumab,nivolumab, or pembrolizumab; (b) an antibody specific for PD-L1, such asMDX-1105, BMS-936559, MEDI4736, MPDL3280A, or MSB0010718C; or (c) anantibody specific for CTLA4, such as tremelimumab or ipilimumab.

In further embodiments, this disclosure provides methods of treating adisease or disorder associated with CD20 expression, comprisingadministering to a subject having or suspected of having a disease ordisorder associated with CD20 expression a therapeutically effectiveamount of a host cell comprising a heterologous nucleic acid moleculeencoding a fusion protein that comprises or consists of an amino acidsequence of any one of SEQ ID NOS.:26-29, 32-38, and 41-43, and atherapeutically effective amount of an immune checkpoint inhibitor,optionally wherein the immune checkpoint inhibitor is selected from (a)an antibody specific for PD-1, such as pidilizumab, nivolumab, orpembrolizumab; (b) an antibody specific for PD-L1, such as MDX-1105,BMS-936559, MEDI4736, MPDL3280A, or MSB0010718C; or (c) an antibodyspecific for CTLA4, such as tremelimumab or ipilimumab.

Exemplary chemotherapeutic agents include alkylating agents (e.g.,cisplatin, oxaliplatin, carboplatin, busulfan, nitrosoureas, nitrogenmustards such as bendamustine, uramustine, temozolomide),antimetabolites (e.g., aminopterin, methotrexate, mercaptopurine,fluorouracil, cytarabine, gemcitabine), taxanes (e.g., paclitaxel,nab-paclitaxel, docetaxel), anthracyclines (e.g., doxorubicin,daunorubicin, epirubicin, idaruicin, mitoxantrone, valrubicin),bleomycin, mytomycin, actinomycin, hydroxyurea, topoisomerase inhibitors(e.g., camptothecin, topotecan, irinotecan, etoposide, teniposide),monoclonal antibodies (e.g., ipilimumab, pembrolizumab, nivolumab,avelumab, alemtuzumab, bevacizumab, cetuximab, gemtuzumab, panitumumab,rituximab, tositumomab, trastuzumab), vinca alkaloids (e.g.,vincristine, vinblastine, vindesine, vinorelbine), cyclophosphamide,prednisone, leucovorin, oxaliplatin, hyalurodinases, or any combinationthereof. In certain embodiments, a chemotherapeutic is vemurafenib,dabrafenib, trametinib, cobimetinib, sunitinib, erlotinib, paclitaxel,docetaxel, or any combination thereof. In some embodiments, a patient isfirst treated with a chemotherapeutic agent that inhibits or destroysother immune cells followed by a pharmaceutical composition describedherein. In some cases, chemotherapy may be avoided entirely.

In any of the embodiments described herein, the methods of thisdisclosure are applied to a subject that has been pre-treated with aCD20-specific binding molecule, optionally wherein the CD20-specificbinding molecule is rituximab, ofatumumab, ocrelizumab, ublituximab,veltuzumab, or any combination thereof; or a chemotherapeutic (e.g., aCHOP [cyclophosphamide-Hydroxydaunorubicin-Oncovin-Prednisone], CHOP-R[R is rituximab], or CHOEP or CHOEP-R [E is etoposide] regimen); or aninhibitor of an immune suppression component (e.g., an antibody againstPD-1, PD-L1, CTLA4, or the like).

Administration of certain compounds of this disclosure (e.g. antibodies,chemotherapeutic agents or immune modulators), or their pharmaceuticallyacceptable salts, in pure form or in an appropriate pharmaceuticalcomposition, can be carried out using any mode of administration foragents serving similar utilities. The pharmaceutical compositions ofthis disclosure can be prepared by combining a compound of thisdisclosure with an appropriate pharmaceutically acceptable carrier,diluent or excipient, and may be formulated into preparations in solid,semi solid, liquid or gaseous forms, such as tablets, capsules, powders,granules, ointments, solutions, suppositories, injections, inhalants,gels, microspheres, and aerosols. Exemplary routes of administering suchpharmaceutical compositions include oral, topical, transdermal,inhalation, parenteral, sublingual, buccal, rectal, vaginal, andintranasal.

The term “parenteral” as used herein includes subcutaneous injections,intravenous, intramuscular, intrasternal injection or infusiontechniques. Pharmaceutical compositions of this disclosure (e.g.,chemotherapeutic agents or immune modulators) are formulated to allowthe active ingredients contained therein to be bioavailable uponadministration of the composition to a patient. Compositions that willbe administered to a subject or patient take the form of one or moredosage units, where for example, a tablet may be a single dosage unit,and a container of a compound of this disclosure in aerosol form mayhold a plurality of dosage units. Actual methods of preparing suchdosage forms are known, or will be apparent, to those skilled in thisart (see, e.g., Remington: The Science and Practice of Pharmacy, 22ndEdition (Pharmaceutical Press, 2012). The composition to be administeredwill, in any event, contain a therapeutically effective amount of acompound of this disclosure, or a pharmaceutically acceptable saltthereof, for therapeutic methods in accordance with the teachings ofthis disclosure.

As a solid composition for oral administration, the pharmaceuticalcomposition may be formulated into a powder, granule, compressed tablet,pill, capsule, chewing gum, wafer or the like form. Exemplary solidcompositions can contain one or more inert diluents or edible carriers.In addition, one or more additives may be present, including binderssuch as carboxymethylcellulose, ethyl cellulose, microcrystallinecellulose, gum tragacanth or gelatin; excipients such as starch, lactoseor dextrins, disintegrating agents such as alginic acid, sodiumalginate, Primogel, corn starch and the like; lubricants such asmagnesium stearate or Sterotex; glidants such as colloidal silicondioxide; sweetening agents such as sucrose or saccharin; a flavoringagent such as peppermint, methyl salicylate or orange flavoring; or acoloring agent. When a pharmaceutical composition is in the form of acapsule, such as a gelatin capsule, it may contain, in addition tomaterials of the above type, a liquid carrier such as polyethyleneglycol or oil or combinations thereof.

The pharmaceutical composition may be in the form of a liquid, such asan elixir, syrup, solution, emulsion, or suspension. In certainembodiments, a liquid composition may be formulated for oraladministration or for delivery by injection, as two examples. Whenintended for oral administration, exemplary compositions may furthercontain, in addition to one or more compounds of this disclosure, asweetening agent, preservative, dye/colorant, flavor enhancer, or anycombination thereof. Exemplary compositions intended for administrationby injection may further contain a surfactant, preservative, wettingagent, dispersing agent, suspending agent, buffer, stabilizer, isotonicagent, or any combination thereof.

Liquid pharmaceutical compositions of this disclosure, whether they aresolutions, suspensions or other like forms, may further compriseadjuvants, including sterile diluents such as water for injection,saline solution, preferably physiological saline, Ringer's solution,isotonic sodium chloride, fixed oils such as synthetic mono ordiglycerides which may serve as the solvent or suspending medium,polyethylene glycols, glycerin, propylene glycol or other solvents;antibacterial agents such as benzyl alcohol or methyl paraben;antioxidants such as ascorbic acid or sodium bisulfite; chelating agentssuch as ethylenediaminetetraacetic acid (EDTA); buffers such asacetates, citrates or phosphates and agents for the adjustment oftonicity such as sodium chloride or dextrose. The parenteral preparationcan be enclosed in ampoules, disposable syringes or multiple dose vialsmade of glass or plastic. Physiological saline is a preferred adjuvant.An injectable pharmaceutical composition is preferably sterile.

A pharmaceutical composition of this disclosure may be intended fortopical administration, in which case the carrier may comprise asuitable solution, emulsion, ointment, gel base, or any combinationthereof. The base, for example, may comprise petrolatum, lanolin,polyethylene glycols, bee wax, mineral oil, diluents such as water andalcohol, emulsifiers, stabilizers, or any combination thereof.Thickening agents may be present in a pharmaceutical composition of thisdisclosure for topical administration. If intended for transdermaladministration, the composition may include a transdermal patch oriontophoresis device.

A pharmaceutical composition of this disclosure may be intended forrectal administration, in the form, for example, of a suppository, whichwill melt in the rectum and release the active compound(s). Acomposition for rectal administration may contain an oleaginous base asa suitable nonirritating excipient. Exemplary bases include lanolin,cocoa butter, polyethylene glycol, or any combination thereof.

A pharmaceutical composition of this disclosure may include variousmaterials that modify the physical form of a solid or liquid dosageunit. For example, a composition may include materials that form acoating shell around the active ingredient(s). Exemplary materials forforming a coating shell may be inert, such as sugar, shellac, or otherenteric coating agents. Alternatively, active ingredient(s) may beencased in a gelatin capsule.

In certain embodiments, compounds and compositions of this disclosuremay be in the form of a solid or liquid. Exemplary solid or liquidformulations include semi solid, semi liquid, suspension, and gel forms.A pharmaceutical composition of this disclosure in solid or liquid formmay further include an agent that binds to the compound of thisdisclosure and thereby assists in the delivery of the compound. Suitableagents that may act in this capacity include a monoclonal or polyclonalantibody, a protein, or a liposome.

A pharmaceutical composition of this disclosure may consist of dosageunits that can be administered as an aerosol. The term aerosol is usedto denote a variety of systems ranging from those of colloidal nature tosystems consisting of pressurized packages. Delivery may be by aliquefied or compressed gas or by a suitable pump system that dispensesthe active ingredients. Aerosols of compounds of this disclosure may bedelivered in single phase, bi phasic, or tri phasic systems in order todeliver the active ingredient(s). Delivery of the aerosol includes thenecessary container, activators, valves, subcontainers, and the like,which together may form a kit.

Pharmaceutical compositions of this disclosure may be prepared bymethodology well known in the pharmaceutical art. For example, apharmaceutical composition intended to be administered by injection canbe prepared by combining a compound of this disclosure with sterile,distilled water to form a solution. A surfactant may be added tofacilitate the formation of a homogeneous solution or suspension.Surfactants are compounds that non covalently interact with the compoundof this disclosure to facilitate dissolution or homogeneous suspensionof a compound in an aqueous delivery system.

Compounds of this disclosure, or their pharmaceutically acceptablesalts, are administered in a therapeutically effective amount, whichwill vary depending upon a variety of factors including the activity ofthe specific compound employed; the metabolic stability and length ofaction of the compound; the age, body weight, general health, sex, anddiet of the patient; the mode and time of administration; the rate ofexcretion; the drug combination; the severity of the particular disorderor condition; and the subject undergoing therapy. Followingadministration of therapies according to the formulations and methods ofthis disclosure, test subjects will exhibit about a 10% up to about a99% reduction in one or more symptoms associated with the disease ordisorder being treated, as compared to placebo-treated or other suitablecontrol subjects.

Compounds of this disclosure, or pharmaceutically acceptable derivativesthereof, may also be administered simultaneously with, prior to, orafter administration of one or more other therapeutic agents. Suchcombination therapy includes administration of a single pharmaceuticaldosage formulation which contains a compound of this disclosure and oneor more additional active agents, as well as administration of thecompound of this disclosure and each active agent in its own separatepharmaceutical dosage formulation. For example, a compound of thisdisclosure and the other active agent can be administered to the patienttogether in a single oral dosage composition such as a tablet orcapsule, or each agent administered in separate oral dosageformulations. Where separate dosage formulations are used, the compoundsof this disclosure and one or more additional active agents can beadministered at essentially the same time, i.e., concurrently, or atseparately staggered times, i.e., sequentially; combination therapy isunderstood to include all these regimens.

It will also be appreciated by those skilled in the art that in theprocess described herein the functional groups of intermediate compoundsmay need to be protected by suitable protecting groups. Such functionalgroups include hydroxy, amino, mercapto, and carboxylic acid. Suitableprotecting groups for hydroxy include trialkylsilyl or diarylalkylsilyl(for example, t-butyldimethylsilyl, t-butyldiphenylsilyl ortrimethylsilyl), tetrahydropyranyl, benzyl, and the like. Suitableprotecting groups for amino, amidino and guanidino includet-butoxycarbonyl, benzyloxycarbonyl, or the like. Suitable protectinggroups for mercapto include C(O) R″ (where R″ is alkyl, aryl orarylalkyl), p methoxybenzyl, trityl or the like. Suitable protectinggroups for carboxylic acid include alkyl, aryl or arylalkyl esters.Protecting groups may be added or removed in accordance with standardtechniques, which are known to one skilled in the art and as describedherein. The use of protecting groups is described in detail in Green, T.W. and P. G. M. Wutz, Protective Groups in Organic Synthesis (1999), 3rdEd., Wiley. As one of skill in the art would appreciate, the protectinggroup may also be a polymer resin such as a Wang resin, Rink resin or a2-chlorotrityl-chloride resin.

It will also be appreciated by those of skill in the art, although suchprotected derivatives of compounds of this disclosure may not possesspharmacological activity as such, they may be administered to a mammaland thereafter metabolized in the body to form compounds of thisdisclosure which are pharmacologically active. Such derivatives may,therefore, be described as “prodrugs”. In certain embodiments, compoundsof this disclosure are in the form of a prodrug.

Furthermore, all compounds of this disclosure that exist in free base oracid form can be converted to their pharmaceutically acceptable salts bytreatment with the appropriate inorganic or organic base or acid bymethods known to those skilled in the art. Salts of the compounds ofthis disclosure can be converted to their free base or acid form bystandard techniques.

In the case of transformed host cells expressing a fusion proteinaccording to this disclosure, administration may be performed usingindividual aliquots of the cells. In certain embodiments, transformedhost cells comprise T cells, which may comprise CD4⁺ T cells, CD8⁺ Tcells, or both. In certain embodiments, T cells comprise a heterologousnucleic acid encoding a chimeric antigen receptor (CAR). In certainembodiments, T cells are sorted to provide for a 1:1 ratio of CD4⁺ andCD8⁺ CD20 CAR T cells for administration to the subject. Cells may beadministered intravenously over approximately 20-30 minutes at thespecified cell dose for each subject. Specified cell doses may bedetermined by the expression level of a transduction marker that isexpressed coordinately with the fusion protein in the vector. Forexample, in certain embodiments, a T cell is transformed using one ormore vectors that coordinately express a truncated CD19 transductionmarker and a CAR. Exemplary CD20 CAR T cell dosage levels for use invarious embodiments of the present disclosure are set forth in Table 1below.

TABLE 1 CD20 CAR T cell formulation and infusion tCD19⁺ CD4⁺/ TotaltCD19⁺ T Dose Level tCD19⁺ CD8⁺ ratio cell dose*^(,)** 0 1:1 1 × 10⁵/kg1 1:1 3.3 × 10⁵/kg  2 1:1 1 × 10⁶/kg 3 1:1 3.3 × 10⁶/kg  4 1:1 1 ×10⁷/kg *per kg recipient weight; **upper limit per dosing level ±15%

In certain embodiments, cells are manufactured from an autologousperipheral blood mononuclear cell (PBMC) product obtained by standardnon-mobilized leukapheresis from the subject. Immunomagnetic selectionmay be performed to enrich CD8⁺ cells or CD4⁺ T cells. In certainembodiments, CD8⁺ cells and CD4⁺ T cells are enriched separately, andeach subset is separately stimulated with, e.g., anti-CD3/CD28paramagnetic beads, followed by transduction with a vector (e.g., alentiviral vector) encoding the fusion protein and, optionally, atransduction marker such as, for example, a tCD19 transduction marker.The transduced T cells may be expanded, then re-stimulated with aCD20-expressing target cell line to boost growth, further expanded exvivo, and then formulated to achieve the specified cell dose forinfusion. For example, in certain embodiments, anti-CD20 CART cells(e.g., 1.5.3-NQ-28-BB-z) according to the present disclosure may bemanufactured in accordance with a method comprising:

-   -   1. Enrichment of CD4⁺ T cells from a fraction of leukapheresis        product or peripheral blood mononuclear cells (PBMC) from whole        blood.    -   2. In parallel with CD4⁺ T cell enrichment, enrichment of CD8⁺ T        cells from the remaining leukapheresis product or PBMC.    -   3. Stimulation of the enriched CD4⁺ and CD8⁺ cells in separate        cultures with clinical grade anti-CD3 and anti-CD28 coated        paramagnetic beads (anti-CD3/CD28 beads) in RPMI 1640 medium        supplemented with glutamine, β mercaptoethanol, and fetal bovine        serum (CTL Media+10% FBS), 50 IU/mL IL-2.    -   4. Transduction of the CD4⁺ and CD8⁺ cells with 1.5.3-NQ-28-BB-z        CAR lentiviral vector on day 1 after anti-CD3/CD28 bead        stimulation.    -   5. Expansion of transduced CD4⁺ and CD8⁺ T cells in CTL        Media+10% FBS and 50 IU/mL IL-2.    -   6. 2× removal of the anti-CD3/CD28 beads by magnetic depletion        on day 4 after CD3/CD28 stimulation.    -   7. Stimulation with an irradiated, clinically qualified,        transformed CD20+ B cell line (TM-LCL) on day 7 after        anti-CD3/CD28 stimulation. This step may be omitted if        in-process cell counts on day 7 predict sufficient cell        expansion without the TM-LCL stimulation.    -   8. Expansion of CD4⁺ and CD8⁺ cells in G-Rex flasks with CTL        Media+10% FBS and 50 IU/mL IL-2.    -   9. Cell harvest of each subset on day 15 (range 13-17) after        anti-CD3/CD28 stimulation, and formulation of a combined        CD4⁺/CD8⁺ T cell product for cryopreservation or infusion.    -   10. Sample collection at appropriate points during the        manufacturing procedure from each of the CD8⁺ and CD4⁺ T cells        for in-process and final release testing.    -   11. Administration to the patient by intravenous infusion at the        indicated dose in 1:1 ratio of tCD19⁺ CD4⁺ and tCD19⁺ CD8⁺ T        cells. Subjects will be pre-treated with lymphodepletion        chemotherapy and receive the T cell infusions at least 36 hours        after completing chemotherapy.

In certain embodiments, cells generated for a subject may be given asfresh cells immediately after manufacture, or may be first cryopreservedand stored in a liquid nitrogen freezer, and then the thawed cellswashed to remove residual cryoprotectant and then formulated forinfusion. The total number of cells will be sufficient to account forcell loss during recovery from thaw and to achieve the cell dose levelspecified in the clinical protocol. In certain embodiments comprisingboth CD4⁺ and CD8⁺ T cells, the total ratio of CD4⁺ and CD8⁺ T cells maydiffer from 1:1 due to differences in transduction of the individualsubsets in individual subjects. For this reason, the subsets may betransduced separately to achieve a desired formulation of the transducedT cells. CD4 and CD8 CAR T cells have demonstrated synergistic effectsin animal models (Sommermeyer et al., Leukemia 2015).

Transformed cells may be suspended in an appropriate cryopreservationmedium (e.g., CryoStor CS10®) for cryopreservation in a controlled ratefreezer. Cryopreserved cells may be stored in the vapor phase of aliquid nitrogen freezer. The fresh or thawed cells may then beresuspended in Normosol+1% HSA and transferred to a transfer pack at thetotal cell dose level specified in the clinical protocol. The formulatedproduct may be stored at 2-8° C. and then transferred under appropriateconditions to the clinical site for administration.

Following leukapharesis, subjects may receive cytoreductive chemotherapyto control disease during production of the transformed cells. Forexample, in certain embodiments, a subject may receive may receivelow-intensity chemotherapy (e.g. lenalidomide, ibrutinib) afterleukapheresis. Prior to administering transformed cells according to thepresent disclosure, chemotherapy or immune modulatory therapy may beappropriate in order to provide lymphodepletion to facilitate survivalof transferred T cells, and to reduce the tumor burden prior to infusionof the cells. For example, subjects may receive lymphodepletingchemotherapy for a predetermined time prior to (e.g., 36-96 hours) theinfusion of the cells. In certain embodiments, a subject may initiallybe treated with a single dose of a chemotherapy agent such ascyclophosphamide (CY) i.v. (e.g., at 1 g/m²) initially. However, if thesubject response rate is determined to be inadequate, thelymphodepletion regimen may be changed so that subsequent patientsreceive a second, further chemotherapeutic or immunomodulatory agent(e.g., CY+fludarabine). Additionally, a subject may, but need not,receive a premedication prior to administration of the cells.

One or more intravenous infusions of the cells described herein may beadministered to the subject following completion of lymphodepletingchemotherapy (e.g., 36-96 hours thereafter). The dose of cellsadministered to the subject may be determined according to the doselevels shown in Table 1, and may be adjusted thereafter to increase,decrease, or otherwise change the amount, composition, ratio, or rate ofthe cells administered. In certain embodiments, a single infusion isadministered to the subject. In further embodiments, a second infusionmay be given if the first infusion does not produce a complete response(CR), or if the disease relapses after a CR. In still furtherembodiments, a third, fourth, fifth, sixth, seventh, eighth, ninth,tenth, or further infusion may be given. In certain embodiments, a cellinfusion may be administered intravenously over a selected period oftime (e.g., approximately 20-30 minutes), adjusted as needed to complywith guidelines for endotoxin limits for parenteral drugs (£ 5EU/kg/hour). The infusion rate may also be adjusted if subjectsexperience mild infusion-related adverse events (grade 2 or lower).

EXAMPLES Example 1 Materials and Methods

Cell Lines

Raji, Daudi, and Ramos (Burkitt lymphoma), Rec-1 (mantle cell lymphoma),and K562 (CD20-negative erythroid leukemia) tumor cell lines wereobtained from ATCC. Granta-519 (mantle cell lymphoma) was obtained fromDSMZ, and FL-18 (transformed follicular lymphoma) was obtained from Dr.David Maloney (Fred Hutchinson Cancer Research Center). CD20 expressionwas authenticated by flow cytometry on all cell lines prior toexperiments. Cell lines were cultured in RPMI 1640 with 25 mM HEPES, 10%fetal bovine serum (FBS), 1% penicillin/streptomycin, and 1% L-glutamineand incubated at 37° C. in 5% CO₂. K562 cells were transduced with aretroviral vector to express CD20, and some cells were again transducedwith a lentiviral vector to express human CD80. Low, medium, and highCD20-expressing K562-CD80 cell lines were obtained by selection afterlimiting dilution cloning. Raji-ffLuc cells were produced bytransduction of Raji cells with retrovirus encoding fireflyluciferase-Thy1.1-Neo and selected with G418 as previously described(James et al., Blood 2009; 114(27):5454-63). Rituximab-refractoryRaji-ffLuc cells were generated with repeated, intermittent cycles ofescalating rituximab concentrations as previously described (Czuczman etal., Clin Cancer Res 2008; 14(5):1561-70).

Flow Cytometry

Ramos cell lines were incubated with rituximab concentrations rangingfrom 0 to 200 μg/ml at room temperature for 30 minutes. Following CD20blocking, anti-CD20-PE antibody (clone L27 [Leu16], BD Biosciences) wasadded, and cells were incubated at either 4° C. or 37° C. for 30minutes. Cells were washed with cold FACS buffer (0.5% fetal bovineserum and 2.5 mM EDTA in PBS) and analyzed on a BD Canto 2 flowcytometer. Data were analyzed using FlowJo version 7.6.1 (TreeStar). Ina separate experiment, FL-18 cells were blocked with varyingconcentrations of rituximab, washed once with FACS buffer, and thenanti-CD20-FITC antibody (clone 1F5, produced in-house from a hybridoma;Press et al., Blood 1987; 69(2):584-91) was added and incubated withblocked cells for 15 minutes at 4° C. Cells were then washed andanalyzed as described above. Similar experiments were also conductedusing ofatumumab instead of rituximab.

Vector Constructs

The CD20-specific Leu16-28-BB-z-tEGFR construct (SEQ ID NO.:57) wasgenerated by amplifying the Leu16 scFv (Wang et al., Hum Gene Ther 2007;18(8):712-25; Wang et al., Mol Ther 2004; 9(4):577-86) by PCR andcloning into NheI and RsrII sites of an epHIV7 lentiviral vectorencoding an IgG4-Fc, CD28, and 41BB domains, and CD3ζ domain (Hudecek etal., Clin Cancer Res 2013; 19(12):3153-64). The Leu16-28-z construct(SEQ ID NO.:49 or 58) was generated by splice overlap PCR of theLeu16-28-BB-z-tEGFR vector to remove the 41BB domain and truncated EGFR.The lentiviral vector encoding the CD20-specific 1F5-28-BB-z CAR hasbeen previously described (Budde et al., PLoS One 2013; 8(12):e82742),but was transferred to the HIV-1-based RRL.sin.cPPT.PGK.GFP.wpreself-inactivating 3^(rd) generation lentiviral vector backbone (Beckeret al., Gene Ther 2010; 17(10):1244-52; from Dr. Hans-Peter Kiem,FHCRC). The Fc spacer region of this construct was modified to abrogatebinding to Fcγ receptors by substituting the IgG1 junction amino acidswith the IgG2 junction amino acids (SEQ ID NO.:9) and adding an N297Qmutation (SEQ ID NO.:10) as previously described (Hudecek et al., Cancerimmunology research 2014; 3(2):125-35; Hombach et al., Gene Ther 2010;17(10):1206-13), to create the 1F5-NQ-28-BB-z construct (SEQ ID NO.:50or 59). To generate the 1.5.3-NQ-28-BB-z CAR construct (SEQ ID NO.:44 or53), a novel scFv sequence was produced by synthesizing the V_(L) andV_(H) sequences from the 1.5.3 fully human anti-CD20 antibody (see,e.g., Bornstein et al., Invest New Drugs 2010; 28(5):561-74; PCTPublication No. WO 2006/130458) using a codon optimization algorithm(GenScript), separated by a 15 amino acid glycine-serine linker (SEQ IDNO.:20), preceded by the GM-CSF signal peptide (SEQ ID NO.:18). Anoverlapping fragment produced by splice overlap PCR was used to replacethe scFv domain of the 1F5-NQ-28-BB-z construct, cloning it intoAgeI/SacII restriction sites. The inducible caspase 9 suicide gene anddownstream 2A sequence (SEQ ID NO.:22 or 23) were removed from thisconstruct by splice overlap PCR. The 1.5.3-NQ-28-z construct (SEQ IDNO.:27) was generated by removing the 41BB domain from 1.5.3-NQ-28-BB-zby splice overlap PCR. All constructs were confirmed by Sangersequencing. Lentivirus was produced using 293T cells transientlytransfected with the described backbone vectors as well as the packagingvectors pCGHP-2, pCMV-Rev2, and pCMV-G, and supernatants containingpackaged lentivirus were concentrated 100-fold by centrifugation.

T Cell Isolation and Transduction

Peripheral blood mononuclear cells (PBMC) were obtained either byapheresis from healthy donors consented under Institutional Review Board(IRB)-approved research protocols at the FHCRC or from used Pallleukocyte filters purchased from the Puget Sound Blood Center. PBMCisolated by centrifugation with Ficoll-Paque density gradient mediumunderwent red blood cell lysis with ammonium-chloride-potassium (ACK)buffer and were cryopreserved in 10% DMSO and 90% FBS. For in vitroexperiments, T cells were negatively selected from thawed PMBC by MACSusing a Pan T cell Isolation Kit II (Miltenyi Biotec). For cytotoxicityexperiments, CD8⁺ T cells were positively selected from healthy donorapheresis PBMC by MACS using anti-CD8 antibody coated beads (MiltenyiBiotec) prior to cryopreservation. For some experiments, central memoryT cells (T_(CM)) were isolated from healthy donor apheresis PBMC priorto cryopreservation by negative selection using an AutoMACS device afterincubation with CliniMACS anti-CD14 and anti-CD45RA beads (MiltenyiBiotec), followed by positive selection with CliniMACS CD62L beads. Inother experiments, CD4 and CD8 cells were enriched by positiveimmunomagnetic selection using anti-CD4 or anti-CD8 beads (MiltenyiBiotec). Selected T cells were stimulated with αCD3/αCD28 Ab-coatedHuman T-Expander Beads (Invitrogen) at a 3:1 bead:T-cell ratio.Activated T cells were spin-transduced (2100 rpm for 60 minutes at 32°C.) the next day with lentiviral vector encoding one of the CD20 CARconstructs (multiplicity of infection of 2-6) plus 4-8 μg/ml polybrene.Transduced T cells were cultured in media containing 50 IU/mlrecombinant human interleukin 2 (rhIL-2) with or without 10 ng/mlrhIL-15 (Miltenyi Biotec), incubated for 4-5 days after stimulationbefore magnetic removal of αCD3/αCD28 beads, and analyzed by flowcytometry to confirm CAR expression. CAR⁺ T cells were used infunctional assays.

For in vivo mouse experiments, T_(CM) or CD4 and CD8-enriched T cellswere thawed, activated, and transduced the next day with concentratedlentiviral supernatant encoding the construct indicated in eachexperiment. CD3/CD28 beads were removed on day 5, cells were expanded in50 IU/mL rhIL-2, restimulated on day 7-10 with irradiated CD20⁺ LCL at a1:1 responder:stimulator ratio, and injected into mice 8-11 days afterrestimulation with LCL.

Proliferation and Cytokine Secretion Assays

T cells (2×10⁵ total cells) stained with 5 μM carboxyfluoresceinsuccinimidyl ester (CFSE) were then co-cultured at 1:1 ratios with tumortarget lines that had been irradiated with 8000-10000 cGy. In rituximabblocking experiments, irradiated target cells were incubated for 30minutes at room temperature with various rituximab concentrations priorto co-incubation with T cells. Supernatant was collected 24 hours afterplating and stored at −20° C. until subsequent cytokine analysis byLuminex assay as previously described (Till et al., Blood 2012;119(17):3940-50) to quantify interferon-gamma (IFN-γ), interleukin-2(IL-2), and TNF-α. After 4-5 days, cells were stained with anti-CD3-APC(BioLegend), and CFSE dilution of CD3-gated lymphocytes as a measure ofproliferation was determined by flow cytometry. Cell size as anothermeasure of activation was determined by flow cytometry using thegeometric mean of the forward scatter (FSC-A) parameter, and subtractingthe cell size of resting T cells. Flow cytometry data were analyzedusing FlowJo software (v7.6.1; Treestar, Ashland, OR). In someexperiments, ofatumumab was substituted for rituximab.

Assessment of cytokine secretion was also determined by intracellularstaining of IFN-γ. CD20 CAR⁺ CD4⁺ or CD8⁺ T cells were co-cultured withirradiated K562 or K562-CD20 cells for 24 hours. For intracellularstaining, cells were fixed, permeabilized with BD Cytofix/Cytoperm kit(BD Biosciences) for 15 minutes on ice. Cells were then stained withanti-IFN-γ (Biolegend) for 1 hour on ice after fixation andpermeabilization. Data were analyzed on BD FACSCanto (BD Biosciences).FlowJo Software was used to analyze the data.

Cytotoxicity Assays

Standard ⁵¹Cr-release assays were performed by co-incubating CD20 CARCD8⁺ T cells with ⁵¹Cr-labeled target cell lines for 4-5 hours aspreviously described. (See, Wang, et al. Hum Gene Ther. 2007;18:712-725). Maximal ⁵¹Cr release was determined by directly measuringthe ⁵¹Cr content of supernatants of labeled cells lysed with 5% IGEPALCA-630. Supernatants were harvested into 96-well Lumaplates, air-driedovernight, and counts were assayed with a TopCount (PerkinElmer).Percent cytotoxicity was calculated by the equation:[Sample−Min_(avg)]/[Max_(avg)−Min_(avg)]*100.

For rituximab blocking experiments, ⁵¹Cr-labeled target cell lines wereincubated at various rituximab concentrations (ranging from 0 to 200μg/mL) for 30 minutes (at double the final concentration during theinitial incubation to yield final concentrations of 10, 25, 50, 100, and200 μg/ml) before addition of CAR⁺CD8⁺ T cells at various effector totarget (E:T) ratios. Cells were cultured in duplicate at 37° C. for 5hours in medium containing heat-inactivated FBS, with ⁵¹Cr-labeledrituximab-blocked target cells in U-bottom 96-well plates. Control wellscontained target cells incubated in rituximab-containing medium withoutT cells (denoted in figures as “0:1” E:T ratio) to exclude thepossibility of rituximab-induced CDC. In some experiments ofatumumab wasused in place of rituximab.

In Vivo Assessment of Rituximab Effect on CAR T Cell Efficacy

Groups of 5-10 NOD.Cg-Prkdc^(scid)Il2rg^(tw1Wjl)/SzJ (NOD/SCID/γ^(−/−)[NSG]) mice 6-10 weeks of age (Jackson Laboratory) were inoculated with5×10⁵ rituximab-resistant Raji-ffLuc or Granta-519 lymphoma cells 2-7days by tail vein. 2-7 days later (as indicated in each experiment), 10⁷CD20 CAR T cells (tCD19⁺) or empty vector T cells were injected by tailvein. In the rituximab blocking experiment, 25 or 200 μg of rituximabwas administered intraperitoneally (i.p.) 5 days after tumor inoculationand 1 day before administration of CAR T cells. Bioluminescence imagingto determine tumor growth was performed using known methods (see, Jameset al., Blood 2009; 114(27):5454-63; Rufener et al., Cancer Immunol Res.2016; 4:509-519). Binning and exposure were adjusted to achieve maximumsensitivity without leading to image saturation. Survival curves weregenerated using the Kaplan-Meier method with GraphPad Prism 6 software.

To test for persistence of adoptively transferred T cells, whole bloodcollected at various timepoints by retro-orbital bleeding was lysed byACK lysing buffer (Quality Biological). Mouse serum was obtained bycentrifugation of clotted blood specimens from the retro-orbital plexuson days 6 and 13 after tumor inoculation, and serum rituximab levelswere measured using an ELISA assay to determine rituximab concentrationsas previously described (see, Gopal A K, et al., Blood 2008;112(3):830-5; Maloney D G, et al., Blood 1997; 90(6):2188-95). Fcreceptors of isolated cells were blocked with intravenous immunoglobulin(IVIG), and cells were stained with monoclonal antibodies (mAbs) tomCD45 (30-F11, Biolegend), hCD3 (HTT3a, Biolegend), and hCD19 (HIB19, BDBioscience). Data were collected with a BD Canto 2 and analyzed onFlowJo Software (Treestar). Mouse studies were approved by the FHCRCInstitutional Animal Care and Use Committee.

Patient Serum Samples

Human serum samples were provided by B-cell lymphoma patients followingIRB approval and informed consent obtained in accordance with theDeclaration of Helsinki. Serum samples were collected within 4 monthsafter rituximab-containing salvage chemoimmunotherapy. Serum rituximabconcentrations were determined as previously published (Maloney et al.,Blood 1997; 90(6):2188-95).

Example 2 Effect of Rituximab on CD20 Binding by CAR ContainingAnti-CD20 scFv

CD20-directed CARs using scFvs derived from two different murinemonoclonal antibodies, either the Leu16 (L27; see, Till et al., Blood2008; 112(6):2261-71; Till et al., Blood 2012; 119(17):3940-50) or 1F5antibodies (see, Wang J, et al., Hum Gene Ther 2007; 18(8):712-25; Buddeet al., PLoS One 2013; 8(12):e82742), each of which bind to epitopes onthe large extracellular loop of the CD20 molecule, were previouslytested (see, Polyak et al., Blood 2002; 99(9):3256-62). These CD20epitopes overlap with the rituximab epitope (see, Polyak et al., Blood2002; 99(9):3256-62) and, thus, rituximab would be expected to block thebinding of these CARs. Using flow cytometry, the ability of varyingconcentrations of rituximab to block binding of the Leu16 anti-CD20antibody to CD20 expressed on Ramos lymphoma cells was assessed bypre-incubating these cells with rituximab prior to incubation with theLeu16 Ab. A dose-dependent blockade of CD20 was observed, with nearcomplete blockade at 50 μg/ml rituximab at 4° C. But, when anti-CD20-PE(Leu16) was incubated at the physiologically relevant temperature of 37°C., low-level CD20 binding occurred even at 200 μg/ml of rituximab(FIGS. 2A-2F). Similar findings were observed in experiments using the1F5 anti-CD20 antibody on FL-18 cells (data not shown). Thus, rituximabbinds to overlapping epitopes with the anti-CD20 CARs of this disclosureand has the potential to interfere with CAR T cell activity againstCD20⁺ target cells.

Example 3 Effect of Rituximab on In Vitro Function of CAR T Cells

The impact of CD20 blocking by rituximab on the function of CD20 CAR Tcells was assessed by measuring proliferation, cytokine secretion, andcytotoxicity using five different CD20 CAR lentiviral constructs afterincubation with a variety of CD20⁺ B cell NHL cell lines. The CARconstructs (FIGS. 1A and 1B) were the 3^(rd)-generationLeu16-28-BB-z-tEGFR and 1F5-28-BBz constructs (see, Budde et al., PLoSOne 2013; 8(12):e82742), the 2^(nd)-generation Leu16-28-z construct, andtwo CD20 CARs (1.5.3-NQ-28-BB-z and 1.5.3-NQ-28-z) derived from thefully human 1.5.3 anti-CD20 Ab, which also binds to an overlappingepitope with rituximab (see, Bornstein et al., Invest New Drugs 2010;28(5):561-74). CAR expression was typically achieved in 40-80% of the Tcells (data not shown).

Proliferation of CFSE-labeled CAR T cells was largely unimpaired whencultured with various NHL target cell lines (Raji, Daudi, Rec-1, andFL-18) in the presence of rituximab. CAR T cells stimulated with targetcells in the presence of rituximab at concentrations up to 200 μg/mlexhibited >96% of the proliferation observed after stimulation in theabsence of rituximab (FIG. 3A). Cell size is another measure of T cellactivation (see, Grumont et al., Immunity 2004; 21(1):19-30). CAR⁺ Tcells were analyzed by flow cytometry for forward scatter as an estimateof cell size and found that following stimulation with Raji, Daudi, orRec-1 tumor cells pre-incubated with rituximab, CART cells exhibited amedian size >85% of the size of control cells not exposed to rituximab(FIG. 3A). T cells incubated with FL-18 cells exhibited a slightly morepronounced, but still modest, reduction in cell size followingincubation with rituximab (73% of control cell size at 200 μg/ml).

In contrast to proliferation, cytokine secretion by CAR T cells wasfound to be decreased in the presence of increasing rituximab levels(FIG. 3B). However, even at 100 μg/ml of rituximab, the cytokines IFN-γ,IL-2, and TNF-α were produced at 34-51%, 70-92%, and 79-108% of baselinelevels, respectively. Similar findings were observed using K562 cellsgenetically modified to express CD80 and CD20 as targets, withCD20-negative K562-CD80 cells as a control to demonstrate antigenspecificity of CD20 CAR T cell activity (FIGS. 6A and 6B).

The impact of rituximab on the cytolytic activity of CAR⁺ T cellsagainst various CD20⁺ NHL target cell lines was also examined. Usingstandard ⁵¹Cr-release assays with CAR⁺/CD8⁺ T cells as effectors andRaji, FL-18, Granta, or Rec-1 as targets, cytotoxicity was found to beminimally impaired at rituximab concentrations up to 50 μg/ml (FIG. 4 ),and >65% of baseline cytolytic activity was retained in rituximabconcentrations of 100 μg/ml against all target cell lines tested.

The in vitro functionality of the fully human 1.5.3-NQ-28-z and1.5.3-NQ-28-BB-z CAR T cells were tested in the presence of rituximab.As with the Leu16 and 1F5 CARs, a modest dose-dependent decrease incytokine secretion and cytotoxicity against rituximab pre-treated targetcells was observed, but not proliferation.

Example 4 Effect of Expression Levels of CD20 on CAR T Cell Sensitivityto Anti-CD20

To examine whether the level of CD20 expression on tumor cells mightimpact sensitivity to rituximab blockade, K562-CD80 cell lines with low,medium, and high levels of CD20 expression after limiting dilutioncloning (FIG. 10 ) were selected for testing. The in vitro CAR T cellfunction was again assessed in the presence of varying concentrations ofrituximab. As with the NHL cell lines, proliferation of CAR T cells wascompletely intact regardless of the expression level of CD20 on targetcells (FIG. 5A). Cell size was undiminished when CD20^(high) cells wereused as targets, although a modest reduction in cell size was found forcells expressing lower levels of CD20. In contrast to proliferation andcell size, cytokine secretion was significantly impaired uponstimulation with CD20^(low) target cells, with IFN-γ, IL-2, and TNF-αlevels as low as 5%, 17%, and 22% of baseline values, respectively, at100-200 μg/ml of rituximab (FIG. 5B; FIG. 11A-11E), whereas T cellsstimulated with CD20^(high) targets retained >75% of baseline activityat rituximab concentrations of 100 μg/ml.

The impact of CD20 antigen density on the rituximab-mediated inhibitionof CAR T cell cytolytic activity is shown in FIG. 5C. T cell killing oftarget cells expressing high levels of CD20 was minimally impacted byrituximab, even at low E:T ratios. However, there was a dose-dependentdecrease in T cell cytotoxicity against CD20^(low) and CD20^(medium)K562-CD80 targets, which was most pronounced at lower effector to target(E:T) ratios. Cytolytic activity against CD20^(low) targets was retainedat 47% of baseline at a 50:1 E:T ratio at 200 μm/ml rituximab, but wasonly 16% of baseline at a 2:1 E:T ratio.

Example 5 In Vivo Anti-Tumor Activity of CD20 CAR T Cells in thePresence of Residual Rituximab

The in vitro experiments above indicated that CD20 CAR T cells retainsignificant functionality against CD20⁺ tumors despite the presence ofmoderate levels of rituximab. To evaluate how these observations wouldtranslate to the in vivo setting, the impact of residual rituximab onCAR T cell activity in a mouse lymphoma model was examined.

By way of background, rituximab as a single agent has significantanti-tumor activity against Raji cells in immunocompromised mousexenograft models (see, Hernandez-Ilizaliturri F J, et al., Clin CancerRes 2003; 9(16 Pt 1):5866-73). To overcome a potential confoundingtherapeutic effect from rituximab in combination therapy experiments, arituximab-refractory Raji cell line (RR-Raji) was generated usingpreviously described methods (see, Czuczman M S, et al., Clin Cancer Res2008; 14(5):1561-70), and CD20 expression was found to be retained inthis cell line (FIG. 12 ).

NSG mice were inoculated i.v. with RR-Raji cells and some groups weretreated with high or low-dose rituximab once tumors were established 5days after inoculation, and then CD20 CAR⁺ T cells were administeredi.v. the following day (FIG. 7A). Mice that received rituximab alonedemonstrated a modest, transient anti-tumor effect, but all died oftumor progression by day 24, whereas mice treated with CAR T cells alonehad significant tumor regression, with tumor eradication in 40% of miceand a doubling of median survival (52 days). Mice that receivedrituximab the day prior to T cell infusion did not have impaired in vivoCAR T cell activity as compared to mice receiving CAR T cells alone; allbut one mouse in the 25 μg/ml rituximab group and all mice in the 200μg/ml rituximab group demonstrated tumor eradication (FIGS. 7B and 7C;FIGS. 13A and 13B).

To confirm that these tumor remissions occurred in the presence ofphysiologically relevant serum levels of rituximab, serum fromrituximab-treated mice was collected on the day of T cell infusion andone week later and serum rituximab levels were measured. Mice receiving200 μg/ml rituximab had an initial median serum rituximab concentrationof 138.5 μg/ml (range 54.5-173.6) and 39.7 μg/ml (range 1.6-51.9) a weeklater, and mice receiving 25 μg/ml rituximab had a median concentrationof 11.7 μg/ml (range 2.8-17.8) at baseline and 0 μg/ml at 1 week after Tcell infusion (FIG. 7D).

In addition, circulating CAR T cell levels were quantified by flowcytometry 28 days after tumor injection. There was no significantdifference CAR T cell levels between mice receiving CAR T cells alone orrituximab plus CAR T cells, indicating that the presence of rituximabdid not impair the in vivo persistence of CAR T cells (FIGS. 14A-14C).

Example 6 Serum Rituximab Concentrations of Patients Treated withSalvage Rituximab-Containing Regimens

To place the above-noted results into a clinical context, a clinicallyrelevant range of residual serum rituximab levels in the intendedpatient population was queried in a database of patients with B-cell NHLwho underwent autologous stem cell transplantation on investigationalprotocols and had a pre-transplant serum rituximab measurement available(see, Gopal et al., Blood 2008; 112(3):830-5). A total of 103 patientswho received a rituximab-containing chemotherapy regimen within 4 monthsof the serum blood draw (range 0.5-3.8 months, median 1.8) wereidentified, and the median rituximab concentration in these patients was38.3 μg/ml, with an interquartile range of 19.1-71.7 μg/ml (FIG. 7E).The rituximab concentration was 100 μg/ml or lower in 86% of patients.

Example 7 Effect of Ofatumumab on CD20 CAR T Cell Function

To determine the importance of epitope location on the effect ofanti-CD20 antibodies on CAR function, the in vitro assays were repeatedwith ofatumumab, an anti-CD20 antibody that binds to a distinct epitopefrom rituximab, which involves a smaller extracellular loop of CD20 aswell as a different area of the large loop (see, Du et al., Mol Immunol2009; 46(11-12):2419-23; Teeling et al., J Immunol 2006; 177(1):362-71).The ability of ofatumumab to block binding of the Leu16 anti-CD20antibody was first evaluated by flow cytometry, which showed thatdespite the different epitope, binding of the second antibody wasprofoundly blocked by ofatumumab. Moreover, the blocking of binding wasat even lower concentrations than rituximab (FIGS. 2D-F). Then in vitrofunctional assays were performed on Rec-1 and Raji-ffLuc lymphoma cellsthat had been pre-incubated with varying concentrations of ofatumumab(FIGS. 8A-8C). The results were similar to those with rituximab, in thatproliferation and cell size were minimally affected, but cytokineproduction was more impacted, in a dose-dependent manner. Compared withrituximab, cytotoxicity was more profoundly impaired in the presence ofofatumumab. These findings indicated that the inhibitory effect ofanti-CD20 antibody is due to steric inhibition and not to directblocking of the CAR binding epitope. Hence, the stronger inhibitoryeffect of ofatumumab resulted from a slower off-rate compared withrituximab. This was supported by competitive cell-binding flow cytometrystudies at 4° C. or 37° C. (FIGS. 2D-F), which confirmed a much lowerdissociation of ofatumumab, consistent with previously reported data(see, Teeling et al., Blood 2004; 104(6):1793-800).

Example 8 Cytokine Secretion by Various CAR Constructs In Vitro

Central memory (CD14⁻CD45RA⁻CD62L⁺) T cells were stimulated withanti-CD3/CD28 antibody coated beads, transduced 24 hours later withlentiviral vectors encoding the indicated CAR constructs, and expandedin vivo. At day 14, the cells were re-stimulated with either irradiatedRaji-ffLuc cells (FIG. 15A and FIG. 15C), Granta-519 cells (FIG. 15B),and Jeko cells (FIG. 15D). The “19-BB-z” construct is a clinical-gradeCD19-targeted CAR being used in clinical trials and is provided as apositive control. Supernatants were harvested 24 hours later andanalyzed by Luminex assay for interferon (IFN)-γ, IL-2, and tumornecrosis factor-α levels.

Example 9 Cytokine Secretion by CD20 CAR T Cells

CD4⁺ and CD8⁺ T cells transduced with the 1.5.3-NQ-28-BB-z lentiviralvector and expanded ex vivo were restimulated with irradiated Raji-ffLucCD20⁺ lymphoma cells. Secretion of the indicated cytokines was measuredin cell supernatants after 24 hours by Luminex assay. (FIG. 16A).Cryopreserved CD4⁺ and CD8⁺ CD20 CAR T cells were thawed andrestimulated with K562 cells or K562 cells expressing CD20 and at 24hours were analyzed by intracellular staining for IFN-γ by flowcytometry. (FIG. 16B).

Example 10 In Vitro Cytotoxicity of Various CAR Constructs

Central memory (CD14⁻CD45RA⁻CD62L⁺) T cells were stimulated withanti-CD3/CD28 antibody coated beads, transduced 24 hours later withlentiviral vectors encoding the indicated CAR constructs, and expandedin vivo. At day 14, the cells were used as effectors in a standard4-hour ⁵¹Cr-release assay, using (FIGS. 17A and 17B) Raji-ffLuc, and(FIG. 17B) Jeko cells as targets. The “19-BB-z” construct is aclinical-grade CD19-targeted CAR being used in clinical trials and isprovided as a positive control. The specific target cell lysis of eachCAR T cell population is shown.

Example 11 Proliferation of CD20 CAR T Cells

CD8⁺ T cells were transduced with the 1.5.3-NQ-28-BB-z lentiviral vector(or were mock-transduced) and expanded ex vivo, and then cryopreserved.The cells were then thawed, stained with carboxyfluorescein succinamidylester (CFSE), and restimulated with irradiated CD20⁺ Raji-ffLuc lymphomacells, K562 cells, or K562 cells expressing CD20. Cells were analyzed byflow cytometry 4 days later. (FIG. 18A) CFSE dilution of CAR⁺ cells(gated on CD3⁺/tCD19⁺) is shown. The dashed-line histogram shows CFSEfluorescence of T cells in culture medium only, and solidline-histograms are T cells co-incubated with target cells. (FIG. 18B)The percentage of divided cells is shown for each group.

Example 12 In Vivo Anti-Tumor Activity of Various CAR Constructs

Central memory (CD14⁻CD45RA⁻CD62L⁺) T cells were stimulated withanti-CD3/CD28 antibody coated beads, transduced 24 hours later withlentiviral vectors encoding the indicated CAR constructs, and expandedin vivo. The “19-BB-z” construct is a clinical-grade CD19-targeted CARbeing used in clinical trials at our center and provided as a benchmarkcontrol. NSG mice were injected i.v. with Raji-ffLuc tumor cells,followed 2 days later by i.v. injection of expanded central memory(CD14⁻CD45RA⁻CD62L⁺) T cells transduced with the 1.5.3-NQ-28-BB-z CAR,1.5.3-NQ-28-z CAR, JCAR-014 (anti-CD19-41BB-ζ), or an empty vector.(FIG. 19A) Tumor burden over time as assessed by bioluminescenceimaging; and (FIG. 19B) Kaplan-Meier plot of overall survival.

Example 13 In Vivo Activity of CD20 CAR T Cells Against Mantle CellLymphoma

CD4⁺ and CD8⁺ CD20 CART cells were transduced with the 1.5.3-NQ-28-BBzCAR and used to treat NSG mice that had been inoculated 7 days earlierwith Granta-ffLuc mantle cell lymphoma cells by tail vein. AKaplan-Meier plot of overall survival is shown in FIG. 20 .

Example 14 In Vivo CAR T Cell Persistence and Related PhysiologicalEffects

Retroorbital blood samples were obtained at serial time points afterinfusion of either CD20 CAR T cells or empty vector tCD19-expressing Tcells in NSG mice bearing Raji-ffLuc disseminated tumors. CD20 CAR Tcells expressing the tCD19 transduction marker were quantified by flowcytometry at each time point as human CD3⁺/mouse CD45-negative/humanCD19⁺ cells. (FIG. 21A) tCD19⁺ T cells at 3 post-infusion time points asa percentage of total nucleated cells in the blood are shown (n=9initially in CAR T cell group). Truncated CD19⁺ cells from an emptyvector mouse are shown for reference. (FIG. 21B) In a separateexperiment, the tCD19⁺ cells from 2 mice in each group (empty vector vsCAR T cells) are shown longitudinally with weekly measurements.

Additionally, mice treated with CD20 CAR T cells were monitored forsigns of toxicity based on weight, general behavior and appearance,physical activity, posture, grooming habits, skin color, presence ofdiarrhea, signs of eye, mouth, or skin inflammation, lethargy, or signsof severe anemia (pale ear pinnae or feet or mucous membranes). No signsof T-cell-related toxicity were observed in any mice over 11 experimentsusing CD20 CAR T cells, with the exception of the finding of xenogeneicgraft-versus-host disease that developed in some mice at latetime-points. This finding occurred both in mice receiving CD20 CAR Tcells as well as in mice receiving empty vector cells and, thus, is notassociated with the CAR vector but rather is a known consequence ofxenogeneic T cell transfer. In each experiment, the weight of each mousewas recorded at least 3 times per week, and was generally stable exceptin mice that experienced terminal tumor progression, in which weightloss occurred over the last few days of life (data not shown).

Finally, blood samples were taken from a subset of mice in twoexperiments to determine physiological function of the animals. In thefirst experiment, mice bearing Granta mantle cell lymphoma tumors weretreated with either untransduced T cells, CD20 CAR T cells that had notbeen restimulated with CD20⁺ TM-LCL cells, or CD20 CAR T cells that hadbeen restimulated with TM-LCL cells (either freshly infused or firstcryopreserved, then thawed and infused). Renal function was measuredusing blood urea nitrogen (BUN) and creatinine, hepatic function wasmeasured using alanine and aspartate aminotransferases (ALT and AST),and marrow function was measured by white blood cell count (WBC),hemoglobin, and platelet count in retroorbital blood samples in treatedmice (data not shown). Compared with untreated mice, no increases in BUNor creatinine or significant changes in hepatic function were seen inmice treated with CAR T cells. Mice treated with CAR T cells that hadnot been restimulated with TM-LCL cells had a drop in WBC compared withuntreated mice, but this was not observed in mice treated with T cellsthat had been restimulated with TM-LCLs. A small drop in hematocrit, butnot hemoglobin, was observed in mice treated with TM-LCL-restimulatedCAR T cells, though this was not seen in mice receiving non-restimulatedCAR T cells. The platelet count increased in mice receivingnon-restimulated CAR T cells, but no significant changes were seen inmice receiving restimulated CAR T cells.

In the second experiment, mice bearing Raji-ffLuc tumors were treatedwith either low-dose (1×10⁶ CAR+ cells/mouse) or high dose (5×10⁶ CAR+cells/mouse), and renal and hepatic function was assessed. A trendtowards higher BUN was seen in the mice receiving T cells, but there wasno change in serum creatinine (data not shown). No elevation of hepatictransaminases was observed.

Example 15 Clinical Study of Anti-CD20 CAR Therapy

A phase I/II study was designed to assess the safety and maximumtolerated dose (MTD) of adoptive T cell therapy with a 1:1 mixture ofautologous CD4+ and CD8+ T cells transduced to express a CD20-specificCAR, 1.5.3-NQ-28-BB-z. The self-inactivating (SIN) lentiviral vectorcarrying this construct used to transduce T cells in this study is a3^(rd)-generation HIV-1-derived lentivirus, which encodes an scFv fromthe 1.5.3 fully human monoclonal antibody that recognizes an epitope inthe large extracellular loop of human CD20, and which is linked to amodified human IgG1 hinge/spacer region, human CD28 transmembrane andintracellular domains, and the human 4-1BB and CD3ζ signaling domains(FIG. 1A). The vector also encodes a non-functional, truncated cellsurface human CD19 (tCD19) separated from the CAR cassette by aself-cleavable E2A element, which facilitates tracking of the CAR Tcells in vivo. The truncation of CD19 shortens the intracellular domainto 19 amino acids, removing all tyrosine residues that serve asphosphorylation sites, but retains the extracellular epitopes recognizedby anti-CD19 antibodies. The tCD19 can also be used as a target forCD19-targeted antibodies or antibody-drug conjugates to eliminate theCAR T cells, for example, in the case of prolonged B cell aplasia.

Previous efforts investigating anti-CD20 CAR T therapies showed somesuccess, but low transfection efficiency (<0.1%) required antibioticselection and prolonged ex vivo growth, resulting in low CAR expressionand T cell exhaustion (see, e.g., Till et al., Blood 119(17):3940-3950,2012; see also Till et al., Blood 112(6):2261-2271, 2008; Wang et al.,J. Clin. Immunol. 155(2):160-75, 2014; da Silva et al., Blood ASH AnnualMeeting Abstracts: Abstract #1851, 2016).

The clinical trial will enroll 30 subjects with B-cell non-Hodgkinlymphoma, including mantle cell, follicular, lymphoplasmacytic, marginalzone, transformed indolent B cell lymphoma (including transformed CLL),or diffuse large B cell lymphoma that has relapsed after a response toat least one prior therapy regimen or is refractory to prior therapy.Critical eligibility criteria include: age 18 years or older (of anygender, race, or ethnicity); measurable disease with evidence of CD20expression; female participants may not be pregnant or breastfeeding;adequate hepatic, renal, pulmonary, cardiac, and hematologic function asdefined in clinical protocol; no active central nervous systemmetastases or past/current clinically relevant central nervous systempathology; no HIV, active uncontrolled infection, or active autoimmunedisease requiring systemic immunosuppressive therapy.

Patients with de novo DLBCL must meet one of the following criteria:

-   -   Biopsy-proven refractory disease after a frontline regimen        containing both an anthracycline and rituximab or other        anti-CD20 antibody (i.e. “primary refractory”), where any        disease recurring within 3 months of completion of the regimen        is considered refractory.    -   Relapsed or refractory disease after at least one of the        following:        -   At least 2 lines of therapy (including at least one with an            anthracycline and anti-CD20 antibody) Autologous stem cell            transplant        -   Allogeneic stem cell transplant

A diagram of the general treatment schema is provided in FIG. 24 , anddiagrammatic representation of the formulation and model ofadministration of the CAR T cells is provided in FIGS. 25A and 25 B.

Leukapheresis will be performed on each patient to obtain peripheralblood mononuclear cells. Patients ineligible for a vein-to-veinapheresis may elect to have a percutaneous central venous catheterplaced to permit this collection. Patients ineligible for apheresis whohave a hematocrit of at least 38% and a total non-malignant (normal)lymphocyte count of >2000/mcl may undergo phlebotomy of 400 ml of bloodto obtain PBMCs necessary for generation of the CAR T cells. Thisapproach would only be taken in patients that would be enrolled at doselevels 0 (1×10⁵ tCD19+ cells/kg), 1 (3.3×10⁵ tCD19⁺ cells/kg) and 2(1×10⁶ tCD19⁺ cells/kg). Participants will undergo tumor biopsy prior toleukapheresis. PET CT may be performed before or after tumor biopsy andleukapheresis, depending on accessibility of lymph node.

CAR T cells are manufactured from an autologous peripheral bloodmononuclear cell (PBMC) product obtained by standard non-mobilizedleukapheresis for each patient. PBMC undergo immunomagnetic selection toenrich CD8+ and CD4+ T cells separately, and each subset is separatelystimulated with anti-CD3/CD28 paramagnetic beads, followed bytransduction with the 1.5.3-NQ-28-BB-t lentiviral vector encoding thefully human 3rd-generation CD20-specific CAR and tCD19 transductionmarker. The transduced T cells are expanded, then re-stimulated with aCD20-expressing target cell line to boost growth, further expanded exvivo, and then formulated in a 1:1 CD4/CD8 ratio to achieve thespecified cell dose for infusion. Cell products may either be infusedfresh, or cryopreserved and then thawed, washed, and infused.

The CD20 CAR T cell product will consist of a 1:1 ratio of tCD19+ CD4⁺and tCD19⁺ CD8⁺ T cells, where tCD19 is a transduction marker that isco-expressed with the CAR and identifies CAR⁺ cells. The CD20 CAR T-cellproduct generated for each patient may be given either as fresh cellsimmediately after manufacture, or may be first cryopreserved and storedin a liquid nitrogen freezer, and then the thawed cells washed to removeresidual cryoprotectant and then formulated for infusion. The totalnumber of cells will be sufficient to account for cell loss duringrecovery from thaw and to achieve the cell dose level specified in theclinical protocol. The total ratio of CD4⁺ and CD8⁺ T cells may differfrom 1:1, because transduction of the individual subsets is similar butnot identical in individual patients. For this reason, the subsets aretransduced separately enabling precise formulation of transduced Tcells. The rationale for this ratio is based on published workdemonstrating synergy between CD4 and CD8 CAR T cells in animal models(Sommermeyer et al., Leukemia 2015) and on our objective of providing auniform cell product to all patients to assist in evaluating toxicityand efficacy, which is difficult if every patient receives a differentcomposition.

The CD20 CAR T cells will be suspended in CryoStor CS10® or otherappropriate cryopreservation medium for cryopreservation in a controlledrate freezer. Cryopreserved cells will be stored in the vapor phase of aliquid nitrogen freezer. The fresh or thawed CD20 CAR T cells will beresuspended in Normosol+1% HSA and transferred to a transfer pack at thetotal cell dose level specified in the clinical protocol. The formulatedproduct will be stored at 2-8° C. and then transferred on refrigeratedgel packs to the clinical site at either the University of Washington orSeattle Cancer Care Alliance for administration. The product will bereleased by the FHCRC Cell Processing Facility. The cell product shouldbe infused into the research participant within 6 hours of formulation.The FHCRC Cell Processing Facility will be responsible for documentingthe dispensation and return (when applicable) of the investigationalproduct.

Patients will receive lymphodepleting chemotherapy 36-96 hours prior tothe infusion of CD20 CAR T cells. There must be at least a 36-hourinterval between the last dose of chemotherapy and the T cell infusion.The goals of administering chemotherapy are to provide lymphodepletionto facilitate survival of transferred T cells, and to reduce the tumorburden prior to infusion of CD20 CAR T cells. As outlined in thestatistical considerations of the protocol, patients will initially betreated with a single dose of cyclophosphamide (CY) i.v. 1 g/m²initially. However, if the response rate is inadequate, thelymphodepletion regimen will be changed so that subsequent patientsreceive CY+ fludarabine.

Prior to receiving CD20 CAR T cells, participants will be assessed toensure they have not developed any pulmonary, cardiovascular, hepatic,renal, or neurologic toxicities prohibited by the protocol; have notdeveloped uncontrolled, active, and serious infection; and have notreceived treatment with other investigational agents within 30 days of Tcell infusion.

Premedications are not required prior to the administration of the CD20CAR T cell product. Standard premedications may be used at thediscretion of investigator.

Each patient will receive a single intravenous infusion of CD20 CAR Tcells 36-96 hours following completion of lymphodepleting chemotherapy.The dose of CD20 CAR T cells administered to each patient will bedetermined according to the statistical design described in the clinicalprotocol. The dose levels are shown in Table 2 below. A second infusionof CD20 CART cells may be given if the first infusion does not produce aCR, or if the disease relapses after a CR. For this purpose, patientsmust meet criteria specified in the clinical protocol below.

Cell administration: A single cell product, combined from individualaliquots of CD4+ and CD8+ CD20 CAR T cells in a 1:1 ratio, will beadministered intravenously over approximately 20-30 minutes at thespecified cell dose for each subject. The specified T cell dose refersto CAR+ T cells determined by the expression of the truncated CD19transduction marker, which is expressed coordinately with the CAR in thevector. Dose levels planned for administration under the proposedprotocol are as follows:

TABLE 2 CD20 CAR T Cell Formulation and Infusion tCD19⁺ CD4⁺/ TotaltCD19⁺ T Dose Level tCD19⁺ CD8⁺ ratio cell dose*^(,)** 0 1:1 1 × 10⁵/kg1 1:1 3.3 × 10⁵/kg  2 1:1 1 × 10⁶/kg 3 1:1 3.3 × 10⁶/kg  4 1:1 1 ×10⁷/kg *per kg recipient weight **upper limit per dosing level, ±15%;Dose level 1 is the starting dose level

All patients will be monitored during each T cell infusion. Vital signs(including oxygen saturation) should be recorded before and during theinfusion and approximately hourly for 2 hours after the infusion. Oxygensaturation should be monitored with continuous pulse oximetry during theT cell infusion and for 2 hours following T cell infusion. Subjects willremain on the cell infusion unit for a minimum of 2 hours followinginfusion, or until resolution of any infusion-related toxicity deemed topose a significant risk to the study subject as an outpatient.

Infusion Rate: Each cell infusion should be administered intravenouslyover approximately 20-30 minutes, adjusted as needed to comply withguidelines for endotoxin limits for parenteral drugs (£ 5 EU/kg/hour).The infusion rate can also be adjusted if subjects experience mildinfusion-related adverse events (grade 2 or lower).

The primary objective of this study is to estimate the maximum tolerateddose (MTD) of CAR T cells. The MTD for these purposes will be defined asa true dose limiting toxicity rate of 25%, where DLT is defined as Grade3 or higher non-hematologic toxicity attributable to the CAR T cellinfusion occurring within 28 days of the infusion, lasting at least 4days, and not responsive to tociluzimab, dexamethasone, or otheranti-inflammatory drugs. A modification of the continual reassessmentmethod (CRM) will be used to estimate the MTD. The modifications includetreating patients in groups of two (rather than one), and allowing amaximum increase of one dose level between groups. Patients will receivea single intravenous infusion of CD20 CAR T cells at one of fourescalating dose levels beginning with dose level 1 for the first groupof two patients. Dose escalation or de-escalation is determined by theCRM algorithm, taking into account the number of patients experiencing aserious toxicity at each dose level (see above).

Treatment of patients in the dose-escalation/de-escalation groups willbe staggered such that a minimum of a 28-day interval following infusionis required between each set of 2 patients before escalating to the nextdose level. These dose levels will be initially evaluated in combinationwith CY alone, evaluating the CR rate to determine if CY alone hassufficient activity or if fludarabine will be added (CY/flu). If anycriteria are met to switch to CY/flu, the CRM will be reinitiatedstarting at one dose level below the interim recommended dose (with CYalone) in combination with CY/flu. The interim recommended dose will bedefined as lower of either the maximum dose evaluated to date or thenext dose that would have been selected based on the mCRM following the8th, 16th, or 20th patient for the 1^(st), 2^(nd) and 3^(rd) interimanalyses. This evaluation will continue to a total of 30 patients. Ifnone of these criteria are met, the CRM approach will continue with CYalone in an additional 10 patients (to reach a total of 30 patients).For patients who receive a second infusion, DLT and efficacy outcomeswill be evaluated based on the dose of their primary infusion.

Patients receiving CD20 CAR T cells may develop serious toxicity due toT cell activation, proliferation, and cytokine secretion after encounterwith tumor antigen. Cytokine release syndrome, macrophage activation,and neurotoxicity may occur and require intensive care support, and willnot be considered DLTs if they are considered due to T cell recognitionof the tumor unless these toxicities are not reversible after 4consecutive days of treatment with corticosteroids and/or tocilizumab.

If there ever exists sufficient evidence to suggest that the trueprobability of treatment-related death by day 100 exceeds 20%(regardless of dose), enrollment of patients will be suspended pending adetailed review by the PI, study monitor, statistician, and DSMB.Sufficient evidence for this purpose will be defined as any observedoutcome whose lower 80% confidence limit exceeds 20%.

Evaluations will also be performed to provide a preliminary assessmentof efficacy. Secondary objectives of the study include an examination ofefficacy (in terms of rate of remissions, progression-free survival, andin vivo persistence of T cells). These analyses will be performed usingpatients treated with all doses combined with the final lymphodepletionregimen (either CY alone or CY/fludarabine), modeling outcomes as afunction of dose. A logistic regression model will be used to evaluatebinary outcomes (CR and CR/PR). A Cox proportional hazards model will beused to evaluate time-to-event outcomes (PFS, OS). No formal statisticalhypotheses will be tested with respect to these endpoints; rather,estimates and associated confidence intervals will be provideddescriptively.

Additional secondary objectives are to evaluate of the duration ofpersistence of adoptively transferred CD20 CAR T cells and the migrationof adoptively transferred CD20 CAR T cells. To evaluate the persistenceof the CAR T cells, the patient-level area under the curve (AUC) will beestimated and the summary statistics of the AUCs will be evaluated.Migration (if CAR T cells are present post treatment), is defined as thepresence of CART cells in the tumor at day 10-16 and, if applicable, theBM at day 28. The association between AUC and migration with clinicaloutcomes will mostly descriptive in nature including graphicalpresentation.

To evaluate the secondary objectives associated with evaluatingbiological causes of treatment resistance, the following analyses willbe performed. A paired t-test will be used to compare the biomarkerprofiles between baseline tumors and post-treatment tumors withappropriate transformation if needed. A logistic regression model willbe used to evaluate the association between baseline biomarker valuesand response. A landmark analysis among patients achieving a CR or PR at1 month, measuring survival times (PFS and OS) from the landmark time,using a Cox proportional hazard regression model to evaluate theassociation of correlates measured at the time of CR/PR for patients inwhom a biopsy is acquired at that time. Models will include values forall patients/dose levels and include a variable for dose level.

The development of endogenous anti-tumor responses and epitope spreadingwill also be assessed in a largely exploratory fashion. Data at eachtime point will be summarized, and with sufficient data, a mixed effectmodel will be used to model time-varying outcomes. Differential geneexpression analysis will be conducted between patients with and withoutdemonstrated epitope spreading to identify the biomarker associated withimmune response.

Patients in the study who failed to achieve a CR, or who achieve acomplete response (CR) but later relapse, who wish to receive a secondinfusion of CD20 CAR T cells may be eligible to do so, provided that asufficient number of CD20 CART cells can be produced and the criterialisted below are met:

-   -   a. There is evidence of persistent disease after the first T        cell infusion, or the tumor relapses after a CR.    -   b. There were no toxicities attributed to the first infusion        that were dose-limiting or required dose de-escalation    -   c. The patient is ≥30 days from the first T cell infusion.    -   d. There are no clinical and/or laboratory exclusion criteria        (Patients who achieved a CR and later relapsed must have a        post-relapse biopsy demonstrating ongoing CD20 expression on the        tumor cells.

Participants will undergo evaluations at screening, prior tolymphodepleting chemotherapy, during T cell infusions, and at intervalsfollowing each T cell infusion. The following data will be obtained forsafety and toxicity assessment, according to the clinical protocol:

-   -   History and physical exam before and at intervals after T cell        infusions.    -   Pulse oximetry before and during the infusion    -   Hematologic, hepatic, renal, and electrolyte blood tests before        and at intervals after the T cell infusion    -   Lab tests evaluating for tumor lysis syndrome, coagulopathy, and        cytokine release syndrome before and at intervals after the T        cell infusion.    -   Toxicity grading according to NCI CTCAE Version 4.0    -   Serum cytokine levels    -   B cell reconstitution    -   Serum immunoglobulin levels    -   Replication competent lentivirus testing    -   Persistence of genetically modified T cells    -   Adverse event reporting

The various embodiments described above can be combined to providefurther embodiments. All of the U.S. patents, U.S. patent applicationpublications, U.S. patent applications, foreign patents, foreign patentapplications and non-patent publications referred to in thisspecification and/or listed in the Application Data Sheet areincorporated herein by reference, in their entirety. Aspects of theembodiments can be modified, if necessary to employ concepts of thevarious patents, applications and publications to provide yet furtherembodiments.

These and other changes can be made to the embodiments in light of theabove-detailed description. In general, in the following claims, theterms used should not be construed to limit the claims to the specificembodiments disclosed in the specification and the claims, but should beconstrued to include all possible embodiments along with the full scopeof equivalents to which such claims are entitled. Accordingly, theclaims are not limited by the disclosure.

What is claimed is:
 1. An isolated polynucleotide that encodes aCD20-specific fusion protein, wherein the encoded fusion proteincomprises the amino acid sequence of SEQ ID NO.:26.
 2. The isolatedpolynucleotide of claim 1, wherein the encoded fusion protein consistsof the amino acid sequence of SEQ ID NO.:26.
 3. The isolatedpolynucleotide of claim 1, wherein a nucleotide sequence encoding SEQ IDNO.:26 is comprised in a nucleotide sequence encoding SEQ ID NO.:35. 4.The isolated polynucleotide of claim 1, wherein the polynucleotidecomprises or consists of the nucleotide sequence of SEQ ID NO.:44 or SEQID NO.:53.
 5. A vector comprising the polynucleotide of claim
 1. 6. Thevector of claim 5, wherein the vector is a lentiviral vector or aretroviral vector.
 7. A vector comprising the polynucleotide of claim 2.8. The vector of claim 7, wherein the vector is a lentiviral vector or aretroviral vector.
 9. A host cell comprising the polynucleotide of claim1 and capable of expressing the encoded fusion protein.
 10. The hostcell of claim 9, wherein the host cell is a T cell or a T cellautologous to a subject.
 11. The host cell of claim 10, wherein the Tcell is a CD8+ T cell, a CD4+ T cell, a bulk population of T cells, asubpopulation of T cells, a naïve T cell, a memory stem T cell, acentral memory T cell, an effector memory T cell, or any combinationthereof.
 12. A host cell comprising the polynucleotide of claim 2 andcapable of expressing the encoded fusion protein.
 13. The host cell ofclaim 12, wherein the host cell is a T cell or a T cell autologous to asubject.
 14. The host cell of claim 13, wherein the T cell is a CD8+ Tcell, a CD4+ T cell, a bulk population of T cells, a subpopulation of Tcells, a naïve T cell, a memory stem T cell, a central memory T cell, aneffector memory T cell, or any combination thereof.
 15. A method oftreating a disease or disorder associated with CD20 expression in asubject having or suspected of having a disease or disorder associatedwith CD20 expression, comprising administering a therapeuticallyeffective amount of a host cell of claim
 10. 16. The method of claim 15,wherein the host cell is a CD8+ T cell, a CD4+ T cell, a CD8+ T cell anda CD4+ T cell, a bulk population of T cells, a subpopulation of T cells,a naïve T cell, a memory stem T cell, a central memory T cell, aneffector memory T cell, or any combination thereof.
 17. The method ofclaim 15, wherein the disorder or disease associated with CD20expression is a B-cell lymphoma or leukemia such as B-cell non-Hodgkinslymphoma (NHL) (including Burkitt's lymphoma, chronic lymphocyticleukemia (CLL), small lymphocytic lymphoma (SLL), diffuse large B-celllymphoma, follicular lymphoma, immunoblastic large cell lymphoma,precursor B-lymphoblastic lymphoma, and mantle cell lymphoma), hairycell leukemia, Waldenström's macroglobulinemia, B-cell pro-lymphocyticleukemia, CD37+ dendritic cell lymphoma, lymphoplasmacytic lymphoma,splenic marginal zone lymphoma, extra-nodal marginal zone B-celllymphoma of mucosa-associated (MALT) lymphoid tissue, nodal marginalzone B-cell lymphoma, mediastinal (thymic) large B-cell lymphoma,intravascular large B-cell lymphoma, and primary effusion lymphoma, adisease characterized by autoantibody production such as idiopathicinflammatory myopathy, rheumatoid arthritis, juvenile rheumatoidarthritis, myasthenia gravis, Grave's disease, type I diabetes mellitus,anti-glomerular basement membrane disease, rapidly progressiveglomerulonephritis, Berger's disease (IgA nephropathy), systemic lupuserythematosus (SLE), Crohn's disease, ulcerative colitis, idiopathicthrombocytopenic purpura (ITP), anti-phospholipid antibody syndrome,neuromyelitis optica, multiple sclerosis, an autoimmune disease,dermatomyositis, polymyositis, a disease characterized by inappropriateT-cell stimulation associated with a B-cell pathway, multiple myeloma,or melanoma.
 18. The method of claim 15, wherein the disease or disorderassociated with CD20 expression is: (i) a cancer; (ii) a cancerassociated with aberrant B cell activity; (iii) Non-Hodgkins lymphoma(NHL); (iv) chronic lymphocytic leukemia (CLL); (v) a leukemia; or (vi)any combination thereof.
 19. A method of treating a disease or disorderassociated with CD20 expression in a subject having or suspected ofhaving a disease or disorder associated with CD20 expression, comprisingadministering a therapeutically effective amount of a host cell of claim13.
 20. The method of claim 19, wherein the host cell is a CD8+ T cell,a CD4+ T cell, a CD8+ T cell and a CD4+ T cell, a bulk population of Tcells, a subpopulation of T cells, a naïve T cell, a memory stem T cell,a central memory T cell, an effector memory T cell, or any combinationthereof.
 21. The method of claim 19, wherein the disorder or diseaseassociated with CD20 expression is a B-cell lymphoma or leukemia such asB-cell non-Hodgkins lymphoma (NHL) (including Burkitt's lymphoma,chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL),diffuse large B-cell lymphoma, follicular lymphoma, immunoblastic largecell lymphoma, precursor B-lymphoblastic lymphoma, and mantle celllymphoma), hairy cell leukemia, Waldenström's macroglobulinemia, B-cellpro-lymphocytic leukemia, CD37+ dendritic cell lymphoma,lymphoplasmacytic lymphoma, splenic marginal zone lymphoma, extra-nodalmarginal zone B-cell lymphoma of mucosa-associated (MALT) lymphoidtissue, nodal marginal zone B-cell lymphoma, mediastinal (thymic) largeB-cell lymphoma, intravascular large B-cell lymphoma, and primaryeffusion lymphoma, a disease characterized by autoantibody productionsuch as idiopathic inflammatory myopathy, rheumatoid arthritis, juvenilerheumatoid arthritis, myasthenia gravis, Grave's disease, type Idiabetes mellitus, anti-glomerular basement membrane disease, rapidlyprogressive glomerulonephritis, Berger's disease (IgA nephropathy),systemic lupus erythematosus (SLE), Crohn's disease, ulcerative colitis,idiopathic thrombocytopenic purpura (ITP), anti-phospholipid antibodysyndrome, neuromyelitis optica, multiple sclerosis, an autoimmunedisease, dermatomyositis, polymyositis, a disease characterized byinappropriate T-cell stimulation associated with a B-cell pathway,multiple myeloma, or melanoma.
 22. The method of claim 19, wherein thedisease or disorder associated with CD20 expression is: (i) a cancer;(ii) a cancer associated with aberrant B cell activity; (iii)Non-Hodgkins lymphoma (NHL); (iv) chronic lymphocytic leukemia (CLL);(v) a leukemia; or (vi) any combination thereof.